KR101317045B1 - Therapeutic human anti-il-1r1 monoclonal antibody - Google Patents

Abstract

The present invention describes antibodies that interact with interleukin-1 receptor type 1 (IL-1R1). The present invention also describes a method for treating an IL-1 mediated disease by administering a pharmaceutically effective amount of an antibody against IL-1R1 and a method for detecting the amount of IL-1R1 in a sample using an antibody against IL-1R1. Doing.

Interleukin-1 receptor type 1 (IL-1R1), IL-1 mediated disease, human anti-IL-1R1 monoclonal antibody, IL-1 receptor accessory protein (IL-1RAcP)

Description

Therapeutic human anti-IL-1R1 monoclonal antibody {THERAPEUTIC HUMAN ANTI-IL-1R1 MONOCLONAL ANTIBODY}

This application claims priority to US Provisional Serial No. 60 / 408,719, filed September 6, 2002, the disclosure of which is incorporated herein by reference.

The present invention relates to antibodies that bind to interleukin-1 receptor type 1 (IL-1R1) protein. The present invention also provides pharmaceutical compositions for treating IL-1 mediated diseases (eg, rheumatoid arthritis, osteoarthritis and other inflammatory symptoms).

Antibody Development

Inflammation is the body's response to damage from mechanical damage, infection or antigenic stimulation. Inflammatory responses often appear pathological. The symptoms are caused when the inflammation persists even after the inflammation is exacerbated, improperly stimulated, or the injurious agent is removed.

Inflammatory responses are mediated, in particular, by cytokines. One of the most potent inflammatory cytokines found to date is interleukin-1 (IL-1). Increased IL-1 signaling results in persistent inflammation associated with several diseases, and IL-1 is believed to be a major mediator in many diseases and medical conditions. These cytokines are produced primarily by (but not exclusively) cells of the macrophage / monocyte lineage and in two forms: IL-1 alpha (IL-1α) and IL-1 beta (IL-1β). Will be.

IL-1 is a heterodimeric consisting of two transmembrane proteins, IL-1 receptor type 1 (IL-1R1) and IL-1 receptor accessory protein (IL-1RAcP). It interacts with receptor complexes to stimulate cellular responses. IL-1 initially binds to IL-1R1; IL-1RAcP is then supplemented with these complexes (Greenfeder et al. 1995, J. Biol. Chem. 270: 13757-13765; Yoon and Dinarello, 1998, J. Immunology 160: 3170-3179; 1998, J by Cullinan et al. Immunology 161: 5614-5620), cell transduction is induced by signal transduction.

Cell-based binding studies indicate that IL-1RAcP stabilizes IL-1R signaling complexes by slowing ligand off-rate (see Wesche et al. 1998, FEBS Letters 429: 303-306). . While the interaction of IL-1 with IL-1R is fully characterized, the interaction of IL-1RAcP with ligand-binding receptors is incompletely defined. Since IL-1RAcP does not have significant affinity for IL-1 or IL-1R1 alone, but has high affinity for complexes, a novel binding site for IL-1RAcP is formed by IL-1 / IL-1R binding. This binding site is due to the IL-1 residue (see Ettorre et al. 1997, Eur. Cytokine Netw. 8: 161-171). The other molecule, IL-1 receptor antagonist (IL-1ra), competes with IL-1α and IL-1β for receptor binding but does not compensate for IL-1RAcP and, consequently, becomes a non-signaling receptor in use. IL-1 activity is additionally balanced by IL-1R type II, a decoy receptor that binds to the ligand, but is not involved in signaling due to the truncated intracellular domain. IL-1ra and IL-1R type II play a role in reducing the damage and duration of IL-1 mediated inflammatory diseases (Wesche et al. 1998, FEBS Letters 429: 303-306; Dripps et al. 1991, J. Biol). Chem. 266: 10331-10336; see Dripps et al. 1991, J. Biol. Chem. 266: 20331-20335).

Interleukin-1 inhibitors are produced from any protein that can specifically prevent the activation of cellular receptors on IL-1, which is due to a number of mechanisms. The mechanisms are responsible for down-regulation of IL-1 production, free IL-1 binding, inhibition of IL-1R and IL-1 binding, inhibition of IL-1R-IL-1RAcP complex formation, or inhibition of IL-1 signaling regulation after receptor binding. It includes. IL-1 inhibitor groups include:

Interleukin-1 receptor antagonists such as IL-1ra (described below);

Anti-IL-1R monoclonal antibodies (e.g., described in published European Patent Application No. EP 623674, which is incorporated herein by reference);

IL-1 binding proteins such as soluble IL-1 receptors (e.g., US Pat. Nos. 5,492,888; 5,488,032; 5,464,937; 5,319,071; and 5,180,812, which are described herein) Cited by reference;

Anti-IL-1 monoclonal antibodies (eg, International Patent Application Publication Nos. WO 9501997, WO 9402627, WO 9006371, US Patent Nos. 4,935,343, EP 364778, EP 267611) And EP 220063, the contents of which are incorporated herein by reference);

IL-1 receptor accessory proteins and antibodies that bind thereto (see, eg, International Patent Application Publication Nos. WO 96/23067 and WO 99/37773, the contents of which are incorporated herein by reference) );

Inhibitors of interleukin-1β converting enzyme (ICE) or caspase I (see, eg, International Patent Application Publication Nos. WO 99/46248, WO 99/47545 and WO 99/47154) Which is hereby incorporated by reference), which can be used to inhibit IL-1β production and secretion;

Interleukin-1β protease inhibitors; And

Other compounds and proteins that block in vivo synthesis or extracellular release of IL-1.

Typical IL-1 inhibitors are described in the following references: US Pat. No. 5,747,444; 5,359,032; 5,359,032; 5,608,035; 5,608,035; 5,843,905; 5,843,905; 5,359,032; 5,359,032; No. 5,866,576; 5,869,660; 5,869,660; 5,869,315; 5,869,315; 5,872,095; 5,872,095; 5,955,480; 5,955,480; And 5,965,564; International Patent Application Publication Nos. WO 98/21957, WO 96/09323, WO 91/17184, WO 96/40907, WO 98/32733, WO 98/42325, WO 98 / 44940, WO 98/47892, WO 98/56377, WO 99/03837, WO 99/06426, WO 99/06042, WO 91/17249, WO 98 / 32733, WO 98/17661, WO 97/08174, WO 95/34326, WO 99/36426 and WO 99/36415; European Patent Application Publication Nos. EP 534978 and EP 89479; And French Patent Application No. FR 2762514. The contents of all references cited above are hereby incorporated by reference.

Interleukin-1 receptor antagonists (IL-1ra) are human proteins that act as natural inhibitors of interleukin-1 and are members of the IL-1 family, including IL-1α and IL-1β. Preferred receptor antagonists (including IL-1ra and variants and derivatives thereof), as well as methods of making and using them, are described in the following references, the contents of which are incorporated herein by reference: US Pat. No. 5,075,222 Arc; International Patent Application Publication No. WO 91/08285; WO 91/17184; WO 92/16221; WO 93/21946; WO 94/06457; WO 94/21275; WO 94/21235; DE 4219626; WO 94/20517; WO 96/22793; WO 97/28828; And WO 99/36541; Austrian patent application No. AU 9173636; And French Patent Application No. FR 2706772. Proteins include glycosylated forms of IL-1 receptor antagonists as well as non-glycosylated forms.

Clearly, three useful forms of IL-1ra and variants thereof are described in US Pat. No. 5,075,222 ("222 patent"). IL-1raα elutes from a mono Q FPLC column in about 52 mM NaCl dissolved in Tris buffer at pH 7.6, has an isoelectric point of approximately 4.8 and has a molecular weight of 22-23 kD. IL-1raβ elutes from a mono Q column at 48 mM NaCl and has the property of a protein having a molecular weight of 22-23 kD. Both IL-1raα and IL-1raβ were glycosylated. IL-1rax is a protein eluting from a mono Q column at 48 mM NaCl, having a molecular weight of 20 kD and having non-glycosylated properties. The “222 patent” also describes a method for isolating a gene responsible for coding an inhibitor, a method for cloning a gene in appropriate vectors and cell types, and a method for expressing a gene to produce an inhibitor. Although effective, IL-1ra has a relatively short half-life. Currently in use IL-1ra is administered once a day. Thus, the field benefits from antagonists of IL-1 receptors that have significantly longer half-lives.

Blocking IL-1 signaling by inhibiting the binding of IL-1 to the IL-1 receptor is an effective therapeutic approach to treat IL-1 mediated diseases. There is a need in the art for clinically effective inhibitors of the IL-1 signaling pathway that can ameliorate the effects of IL-1 mediated diseases, which inhibitors are suitable for transport to human patients. Human antibodies that block IL-1 signaling are particularly advantageous in fulfilling this need and provide longer half-lives than current treatments.

SUMMARY OF THE INVENTION

The present invention provides monoclonal antibodies that bind to interleukin-1 receptor type I (IL-1R1). Preferably, the antibody inhibits IL-1 signaling by competing with IL-1β and IL-1α that bind IL-1R1. The invention also provides a hybridoma cell line which produces the monoclonal antibodies of the invention and most preferably secretes the antibodies into cell culture medium. Antibodies of the invention successfully block IL-1 signaling in human cells and are useful for treating patients suffering from IL-1 mediated diseases. The invention also provides a fusion protein comprising a sequence of an antibody Fc region and one or more sequences selected from: SEQ ID NO: 10, SEQ ID NO: 12, SEQ IN NO: 14, SEQ ID NO: 16 , SEQ ID NO: 18, SEQ IN NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38 and SEQ ID NO: 40. Such molecules may be prepared by methods described in, for example, WO 00/24782, which is incorporated herein by reference. Can be prepared. Such molecules can be expressed, for example, in mammalian cells (eg, Chinese hamster ovary cells) or bacterial cells (eg, E. coli cells).

In certain aspects, the present invention provides antibodies, preferably monoclonal antibodies, most preferably human antibodies, comprising heavy and light chains, wherein the heavy chain is SEQ ID NO: 2, SEQ ID NO: 6 and SEQ Amino acid sequences as described in any of ID NO: 8, or antigen-binding fragments thereof or immunologically functional immunoglobulin fragments.

The present invention also provides antibodies, preferably monoclonal antibodies, most preferably human antibodies, comprising heavy and light chains, wherein the light chain is an amino acid sequence set forth in SEQ ID NO: 4, or antigen binding thereof Fragments or immunologically functional immunoglobulin fragments.

In certain aspects, the antibodies of the invention comprise heavy and light chains, wherein the variable region of the heavy chain comprises the amino acid sequence set forth in any one of SEQ ID NO: 10, SEQ ID NO: 14 and SEQ ID NO: 16, Or antigen binding fragments thereof or immunologically functional immunoglobulin fragments. In other aspects, the variable region of the light chain comprises the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 18, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof. In another aspect, the heavy chain comprises SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 and the amino acid sequence set forth in any one of SEQ ID NO: 36, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment. In another aspect, the light chain comprises the amino acid sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 40, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof. The antibody chain is useful for preparing antibodies that specifically bind to IL-1R1, and the resulting molecules bind to IL-1R1 and / or other target molecules (eg, TNF or TNF receptors). It is also useful for the preparation of bispecific antibodies.

The present invention also provides an antibody that specifically binds IL-1R1, wherein the heavy chain comprises an amino acid sequence as described in SEQ ID NO: 10, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment. A heavy chain variable region comprising a light chain comprising a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 12, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment.

In certain aspects, the invention also provides an antibody comprising a heavy chain and a light chain, wherein the heavy chain is at least 90%, more preferably at least 95% and most preferably about 100% of the amino acid sequence set forth in SEQ ID NO: 10. A first variable region comprising a sequence having 99% identity, wherein the light chain is at least 90%, more preferably at least 95% and most preferably about 99% identity with the amino acid sequence set forth in SEQ ID NO: 12 And a second variable region comprising a sequence having the antibody interacts with IL-1R1.

The present invention also provides an antibody that specifically binds IL-1R1, wherein the heavy chain comprises the amino acid sequence described in SEQ ID NO: 14, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment thereof. A heavy chain variable region comprising a light chain comprising a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 12, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment.

In certain aspects, the invention also provides antibodies comprising heavy and light chains, wherein the heavy chains are at least 90%, more preferably at least 95% and most preferably about amino acids with the amino acid sequence set forth in SEQ ID NO: 14 A first variable region comprising a sequence having 99% identity, wherein the light chain is at least 90%, more preferably at least 95% and most preferably about 99% identity with the amino acid sequence set forth in SEQ ID NO: 12 And a second variable region comprising a sequence having the antibody interacts with IL-1R1.

The invention also provides an antibody that specifically binds IL-1R1, wherein the heavy chain comprises an amino acid sequence as described in SEQ ID NO: 16, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment. A heavy chain variable region comprising a light chain comprising a light chain variable region comprising an amino acid sequence set forth in SEQ ID NO: 18, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment.

In certain aspects, the invention also provides antibodies comprising heavy and light chains, wherein the heavy chains are at least 90%, more preferably at least 95% and most preferably about amino acids with the amino acid sequence set forth in SEQ ID NO: 16. A first variable region comprising a sequence having 99% identity, wherein the light chain is at least 90%, more preferably at least 95% and most preferably about 99% identity with the amino acid sequence set forth in SEQ ID NO: 18 And a second variable region comprising an amino acid sequence having said amino acid, wherein said antibody interacts with IL-1R1.

The present invention also provides an antibody that specifically binds IL-1R1, wherein the heavy chain is SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28 or the amino acid sequence set forth in SEQ ID NO: 30, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof, the light chain comprising an amino acid sequence set forth in SEQ ID NO: 38, or an antigen thereof -Binding fragments or immunologically functional immunoglobulin fragments.

The invention also provides an antibody that specifically binds IL-1R1, wherein the heavy chain is an amino acid sequence as set forth in SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36, or an antigen- Binding fragments or immunologically functional immunoglobulin fragments, and the light chain comprises the amino acid sequence set forth in SEQ ID NO: 40, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment.

The present invention provides embodiments of all of the foregoing, such as single chain antibodies, single chain Fv antibodies, Fab antibodies, Fab 'antibodies and (Fab') ₂ antibodies.

In certain aspects, the invention provides a light chain comprising the amino acid sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 40, or an antigen-binding fragment or immunologically functional immunoglobulin fragment thereof.

In addition, the present invention provides SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO A heavy chain comprising the amino acid sequence set forth in any one of: 34 and SEQ ID NO: 36, or an antigen-binding fragment thereof or an immunologically functional immunoglobulin fragment.

The present invention also relates to an isolated human antibody that specifically binds IL-1R1, wherein the antibody comprises (a) and (b): (a) human heavy chain framework ) Region, human heavy chain CDR1 region, human heavy chain CDR2 region and human heavy chain CDR3 region; And (b) human light chain outline region, human light chain CDR1 region, human light chain CDR2 region and human light chain CDR3 region. In certain aspects, the human heavy chain CDR1 region is a heavy chain CDR1 region of 26F5, 27F2 or 15C4 as shown in FIG. 10 and the human light chain CDR1 region is a light chain CDR1 region of 26F5, 27F2 or 15C4 as shown in FIG. In other aspects, the human heavy chain CDR2 region is a heavy chain CDR2 region of 26F5, 27F2 or 15C4 as shown in FIG. 10 and the human light chain CDR2 region is a light chain CDR2 region of 26F5, 27F2 or 15C4 as shown in FIG. to be. In another aspect, the human heavy chain CDR3 region is a heavy chain CDR3 region of 26F5, 27F2 or 15C4 as shown in FIG. 10 and the human light chain CDR3 region is a light chain CDR3 region of 26F5, 27F2 or 15C4 as shown in FIG. .

The present invention also provides isolated human antibodies that specifically bind to interleukin-1 receptor type 1 (IL-1R1), comprising a human heavy chain CDR1 region, a human heavy chain CDR2 region, and / or a human heavy chain CDR3 region. Wherein the human heavy chain CDR1 has the amino acid sequence of SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63; The human heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66; And / or the human heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69.

The present invention also provides isolated human antibodies that specifically bind to interleukin-1 receptor type 1 (IL-1R1), comprising a human light chain CDR1 region, a human heavy chain CDR2 region, and / or a human heavy chain CDR3 region. Wherein the human light chain CDR1 has the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 71; The human heavy chain CDR2 has the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 73; And / or the human heavy chain CDR3 has the amino acid sequence of SEQ ID NO: 74 or SEQ ID NO: 75.

In certain embodiments, the antibodies of the invention bind to the third domain of IL-1R1, which is shown in FIG. 17. Preferably, the epitope for the antibody of the invention consists of the amino acid sequence YSV, referred to herein as epitope 4 and shown in FIG. 24. The invention also relates to fusion proteins and other molecules capable of binding epitope 4, together with the antibodies mentioned above, collectively referred to herein as "specific binding partners." For example, it will be prepared using the method described in WO 00/24782, which is incorporated by reference. The molecule can be expressed, for example, in mammalian cells (eg, Chinese hamster ovary cells) or bacterial cells (eg E. coli cells).

Moreover, the present invention provides a method for mapping epitopes of selected antigens. In one aspect, the method comprises the following steps: (a) generating a set of fusion proteins, wherein each fusion protein is comprised of (i) an avidin and (ii) an antigen. Including fragments; (b) screening the set of fusion proteins for binding with one or more specific binding partners for the antigen; (c) separating the fusion protein on the medium comprising biotin, whereby the avidin binds to biotin; And (d) analyzing the fusion protein bound by the specific binding partner (s) to determine the binding site of the antigen to the specific binding partner (s). In certain aspects, the specific binding partner is an antibody.

In another embodiment, the present invention comprises administering a pharmaceutically effective amount of a plurality of monoclonal antibodies of the invention, or antigen-binding fragments or immunologically functional immunoglobulin fragments thereof, to an individual in need thereof. To provide a method of treating an IL-1 mediated disease, symptom or disorder.

The invention also provides a method of detecting IL-1R1 concentration in a sample, comprising contacting the biological sample with a monoclonal antibody or antigen-binding fragment thereof of the invention. Anti-IL-1R antibodies of the invention can be used to detect and quantify IL-1R, such as competitive binding assays, direct and indirect sandwich assays, immunoprecipitation assays and enzyme-linked immunosorbent assays (ELISAs). It will be used in all known assay methods (see Sola's 1987, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158, CRC Press, Inc.). The antibody may bind to IL-1R having affinity appropriate for the assay method used.

Preferred specific embodiments of the invention will be apparent from the following detailed description of the preferred embodiments and the claims.

1A-1B show cDNA sequences encoding human anti-IL-1R1 antibody heavy chain IgG1 constant region (SEQ ID NO: 1) (FIG. 1A) and human anti-IL-1R1 antibody heavy chain IgG1 constant region (SEQ ID NO: 2). ) Shows the amino acid sequence (FIG. 1B).

2A-2B show cDNA sequences encoding human anti-IL-1R1 antibody κ chain constant region (SEQ ID NO: 3) (FIG. 2A) and human anti-IL-1R1 antibody κ chain constant region (SEQ ID NO: 4). ) Shows the amino acid sequence (FIG. 2B).

Figures 3A-3B show cDNA sequences encoding human anti-IL-1R1 antibody heavy chain IgG2 constant region (SEQ ID NO: 5) (Figure 3A) and human anti-IL-1R1 antibody heavy chain IgG2 constant region (SEQ ID NO: 6 ) Shows the amino acid sequence (FIG. 3B).

4A-4B show cDNA sequences encoding human anti-IL-1R1 antibody heavy chain IgG4 constant region (SEQ ID NO: 7) (FIG. 4A) and human anti-IL-1R1 antibody heavy chain IgG4 constant region (SEQ ID NO: 8). ) Shows the amino acid sequence (FIG. 4B).

5A-5B show the cDNA sequence (FIG. 5A) encoding the 26F5 anti-IL-1R1 antibody heavy chain variable region (SEQ ID NO: 9) and the 26F5 anti-IL-1R1 antibody heavy chain variable region (SEQ ID NO: 10). The amino acid sequence (FIG. 5B) is shown.

6A-6B show the cDNA sequence (FIG. 6A) encoding the 26F5 anti-IL-1R1 antibody κ chain variable region (SEQ ID NO: 11) and the 26F5 anti-IL-1R1 antibody κ chain variable region (SEQ ID NO: 12 ) Shows the amino acid sequence (FIG. 6B).

7A-7B show the cDNA sequence (FIG. 7A) encoding the 27F2 anti-IL-1R1 antibody heavy chain variable region (SEQ ID NO: 13) and the 27F2 anti-IL-1R1 antibody heavy chain variable region (SEQ ID NO: 14). The amino acid sequence (FIG. 7B) is shown.

8A-8B show the cDNA sequence encoding the 15C4 anti-IL-1R1 antibody heavy chain variable region (SEQ ID NO: 15) (FIG. 8A) and the 15C4 anti-IL-1R1 antibody heavy chain variable region (SEQ ID NO: 16). The amino acid sequence (FIG. 8B) is shown.

9A-9B show the cDNA sequence (FIG. 9A) encoding the 15C4 anti-IL-1R1 antibody κ chain variable region (SEQ ID NO: 17) and the 15C4 anti-IL-1R1 antibody κ chain variable region (SEQ ID NO: 18). ) Shows the amino acid sequence (FIG. 9B).

Figure 10 shows the amino acid sequence arrangement of the anti-IL-1R1 antibody heavy chains named 15C4, 27F2 and 26F5. Complementary decision regions (CDRs) are underlined. CDR1 for 26F5 is shown in SEQ ID NO: 61; CDR1 for 27F2 is shown in SEQ ID NO: 62; CDR1 for 15C4 is shown in SEQ ID NO: 63. CDR2 for 26F5 is shown in SEQ ID NO: 64; CDR2 for 27F2 is shown in SEQ ID NO: 65; CDR2 for 15C4 is shown in SEQ ID NO: 66. CDR3 for 26F5 is shown in SEQ ID NO: 67; CDR3 for 27F2 is shown in SEQ ID NO: 68; CDR3 for 15C4 is shown in SEQ ID NO: 69.

11 shows the amino acid sequence arrangement of the anti-IL-R1-γ antibody light chains named 15C4, 27F2 and 26F5. CDR1 for 26F5 / 27F2 is shown in SEQ ID NO: 70; CDR1 for 15C4 is shown in SEQ ID NO: 71. CDR2 for 26F5 / 27F2 is shown in SEQ ID NO: 72; CDR2 for 15C4 is shown in SEQ ID NO: 73. CDR3 for 26F5 / 27F2 is shown in SEQ ID NO: 74; CDR3 for 15C4 is shown in SEQ ID NO: 74; CDR3 for 15C4 is shown in SEQ ID NO: 75.

12 graphically depicts the inhibitory effect of anti-IL-1R1 antibodies on IL-1R / IL-1β / IL-1RAcP complex formation.

FIG. 13 graphically depicts the inhibitory effect of an anti-IL-1R1 monoclonal antibody, designated 15C4, described herein on IL-1R / IL-1α-IL-1RacP complex formation.

FIG. 14 is a graphical representation of the ability of anti-IL-1R1 antibodies to block IL-1β binding, compared to IgG controls, without significantly interfering with IL-1ra binding.

FIG. 15A is a graphical representation of inhibition of IL-6 production in primary human chondrocytes by anti-IL-1R1 antibodies named 15C4, 26F5 and 27F2 described herein in comparison to IL-1ra.

15B graphically shows the inhibition of IL-6 production in primary human chondrocytes by IL-1ra and monoclonal antibodies 15C4 and 27F2 compared to 10H7 and 24E12, which represent monoclonal antibodies.

FIG. 16 graphically shows the inhibition of IL-6 production in human whole blood by anti-IL-1R1 monoclonal antibodies, designated 15C4, 26F5 and 26F2.

17 shows a human amino acid sequence (SEQ ID NO: 76) and a nucleotide sequence (SEQ ID NO: 77) and a rat nucleotide sequence (SEQ ID NO: 78) and an amino acid sequence (SEQ ID NO:) of a third domain IL-1R1 sequence. 79). The numbered bold lines on the human sequence represent 15 different sites mutated to make up 15 different mutant proteins. Rat residues induced by mutations are described below rat nucleic acid sequences.

18 shows the results of Western blotting analysis showing anti-IL-1R1 monoclonal antibodies recognizing IL-1R1 mutations.

Figure 19 shows (I) activation of the IL-1 signaling pathway, in which IL-1R1 and IL-1β are combined and supplemented with IL-1RacP, and (II) the third, three classes of anti-IL-1R1 antibodies. Domain epitope IL-1 blockers, (III) third domain epitope RAcP blockers, and (IV) first / second domain epitope IL-1 blockers are shown.

20 shows the crystal structures of 15C4 and 27F2 with mutations 10 as described herein. Residues shown in gray represent 15C4 and 27F2 epitopes.

21 shows 15C4 epitopes in the third domain of extracellular IL-1R1.

FIG. 22 shows 24E12 epitopes in the third domain of extracellular IL-1R1. FIG.

Figure 23 shows the amino acid sequence (SEQ ID NO: 59) of the avidin-human IL-1R1-FLAG chimeric protein of the present invention.

FIG. 24 shows amino acid sequence of avidin-cynomolgus IL-1R1-FLAG chimeric protein (SEQ ID NO: 60). Recombinant chicken avidin (listed in italics) is bound to the mature extracellular domain of cynomolgus IL-1R1 (indicated by underlined, bold C-terminal FLAG marker) by a six amino acid linker. The four amino acids of human IL-1R1, introduced alone and in combination with each other into the cynomolgus sequence, are shown in bold under the cynomolgus sequence. Epitope 4 is shown in bold italics and underlined.

25A shows the results of Western blotting analysis of anti-human IL1-R1 antibody (anti-huIL1-R1) that binds to IL-1R1. Antibodies marked with * are used at 5 μg / mL and the remaining antibodies at 1 μg / mL.

25B summarizes the results of the densitometry analysis of two Western blotting experiments.

FIG. 26 shows the results of a multiplexed bead-based binding assay for binding of anti-huIL1R1 antibody to avidin IL-1R1-FLAG protein.

The headings used herein are for configuration purposes only and are not intended to be limiting to the main materials described. All references described in this application are expressly incorporated herein by reference for all purposes.

Justice

When a naturally occurring or experimentally induced disease or medical condition is associated with increasing the level of IL-1 in body fluids or tissues, or when cells or tissues obtained from the human body increase the level of IL-1 in culture, the disease or Medical symptoms are considered "interleukin-1 (IL-1) mediated disease". Increased levels of IL-1 include, for example, excess levels generally measured in particular cells or tissues, or are levels of IL-1 detectable in cells or tissues that generally do not express IL-1. In many cases, IL-1 mediated diseases can also be recognized as two additional symptoms: (1) Diseases that can be emulated experimentally in animals by administering IL-1 or upregulating the expression of IL-1. Or pathological findings related to medical conditions; And (2) a disease derived from an experimental animal model exhibiting a disease or medical condition that can be inhibited or stopped by treatment with an agent that inhibits the action of IL-1. In most IL-1 mediated diseases, at least two of these three types of symptoms are suitable, and in many IL-1 mediated diseases all three symptoms are suitable.

Non-inclusive examples of acute and chronic IL-1-mediated diseases include, but are not limited to: acute pancreatitis; Amyotrophic Lateral Sclerosis (ALS); Alzheimer's disease; Cachexia / anorexia, including AIDS-induced cachexia; Asthma and other lung diseases; Atherosclerosis; Autoimmune vasculitis; Chronic fatigue syndrome; Clostridium related diseases, including Clostridium-related diarrhea; Coronary symptoms and signs, including congestive heart failure, coronary restenosis, myocardial infarction, myocardial dysfunction (eg, sepsis-associated myocardial dysfunction) and coronary artery bypass grafts; Cancers such as multiple myeloma and myeloid leukemia (eg, AML or CML) and other leukemias, as well as tumor metastases; Diabetes (eg, insulin-dependent diabetes); Endometriosis; Fever; Fibromyalgia; Glomerulonephritis; Large host graft disease / transplant rejection; Hemorrhagic shock; Hyperalgesia; Inflammatory bowel disease; Inflammatory symptoms of joints, including osteoarthritis, psoriatic arthritis and rheumatoid arthritis; Inflammatory eye diseases related to, for example, corneal transplantation; Ischemia including cerebral ischemia (eg, brain injury due to trauma, epilepsy, bleeding or seizures that can cause neurodegeneration); Kawasaki disease; Learning disability; Lung disease (eg, ARDS); Multiple sclerosis; Myopathy (eg, muscle protein metabolism, especially in sepsis); Neurotoxicity (eg, HIV induced neurotoxicity); osteoporosis ; Pain, including cancer-related pain; Parkinson's disease; Periodontal periodontal disease; Early delivery; Psoriasis; Reperfusion injury; Sepsis 쇽; Side effects from radiation therapy; Temporomandibular joint disease; Sleep disorder; Uveitis; Or inflammatory symptoms due to sprains, sprains, cartilage damage, trauma, orthopedic surgery, infection, or other disease progression. The methods of the present invention for treating such acute and chronic IL-1 mediated diseases, as well as other IL-1-mediated symptoms and diseases, are described below.

Conventional techniques can be used to prepare recombinant DNA, synthesize oligonucleotides, and perform tissue culture and transformation (eg, electroporation, transfection, and lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly performed in the art or as described herein. The following techniques and methods may be performed according to conventional methods generally known in the art or as described in the general and more specific references described and discussed herein. See, eg, 2001, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., Sambrook et al., Which is incorporated herein by reference for all purposes. See also. Unless otherwise intended to give special meaning, academic terms used in conjunction with the experimental methods and techniques of analytical chemistry, synthetic organic chemistry and pharmaceutical chemistry and pharmaceutical chemistry described herein are well known in the art and generally Used. Standard techniques can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and transport, and treatment of patients.

Except as defined elsewhere, the following terms as used herein are understood to have the following meanings:

The term “isolated polynucleotide” refers to (1) a title nucleotide that does not bind covalently or non-covalently with all or a portion of another polynucleotide that binds the title polynucleotide in its natural state, and (2) a molecule that does not bind in its natural state. A title polynucleotide which binds to or (3) a non-naturally occurring title polynucleotide which binds to all other polynucleotides. Such isolated polynucleotides may be genomic DNA, cDNA, mRNA or other RNA of synthetic origin, or any combination thereof.

As used herein, the term “isolated protein” refers to (1) a title protein in which at least some other proteins are generally found with the title protein, and (2) other proteins of the same origin, e.g. Title protein liberated by (3) the title protein expressed in cells of different species, (4) the title protein isolated from at least about 50% of polynucleotides, lipids, carbohydrates or other substances that bind in nature, (5) Title proteins that are not bound in nature by covalent or non-covalent interactions with portions of the protein that bind to "isolated proteins," (6) covalent or non-covalent interactions with polynucleotides that do not bind in nature. The title protein that is actionably bound, or (7) natural foot The title refers to a protein that is not enemy. Genomic DNA, cDNA, mRNA or other RNA, or combinations thereof of synthetic origin encode such isolated proteins. Preferably, an isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would violate its therapeutic, diagnostic, prophylactic, research, or other uses. .

The term "polypeptide" or "protein" refers to one or more chains of amino acids, each chain comprising amino acids covalently bound by peptide bonds, wherein the polypeptide or protein is a natural protein, ie a naturally occurring cell And a majority of chains that are non-covalently and / or covalently bound by peptide bonds, particularly with sequences of non-recombinant cells, or proteins produced by genetically engineered cells or recombinant cells, It may include a molecule having an amino acid sequence of a natural protein or a molecule in which one or more amino acids of the natural sequence have been deleted, added and / or substituted. The terms “polypeptide” and “protein” in particular include sequences in which one or more amino acids of an anti-IL1-R1 antibody, or anti-ILR-1R1 antibody, have been deleted, added and / or substituted. Thus, a "polypeptide" or "protein" may comprise one ("monomer") or multiple ("multimer") amino acid chains.

The term "polypeptide fragment" refers to a polypeptide that is a monomer or multimer with amino-terminal deletions, carboxy-terminal deletions and / or internal deletions or substitutions of naturally occurring or recombinantly produced polypeptides. In certain embodiments, polypeptide fragments comprise an amino acid chain of at least 5 to about 500 amino acids in length. In certain embodiments, the fragment has at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length. Particularly useful polypeptide fragments include functional domains that include a binding domain. In the case of anti-IL1-R1 antibodies, useful fragments include, but are not limited to: CDR regions, particularly the CDR3 regions of heavy or light chains; Variable domain of a heavy or light chain; A variable region comprising a portion or two CDRs of an antibody chain, and the like.

As used herein, the term "immunologically functional immunoglobulin fragment" means a polypeptide fragment comprising at least the variable domains of an immunoglobulin heavy and light chain. The immunologically functional immunoglobulin fragments of the present invention can bind to ligands, interfere with binding of ligands to their receptors, interfere with biological responses due to binding of receptors to ligands, or all of these. The action can be carried out in combination. Preferably, the immunologically functional immunoglobulin fragments of the invention specifically bind to 1L-1R1.

The terms "naturally occurring" and "natural" refer to biological substances (molecules, sequences, protein complexes, cells, etc.) found naturally and not produced by humans. For example, polypeptides or polynucleotide sequences present in organisms (including viruses) that can be isolated from natural sources and not intentionally modified by humans occur naturally. Likewise, the term "non-naturally occurring" or "non-natural" means a material that is not found naturally or is structurally modified or synthesized by humans.

The term "practically combined" means associated components that enable them to perform their inherent functions under suitable conditions. For example, a regulatory sequence is "operably linked" to a protein coding sequence to express the protein coding sequence under conditions suitable for the transcriptional activity of the regulatory sequence.

The term "regulatory sequence" refers to the title polynucleotide sequence that can affect the expression and processing of the coding sequence to which the regulatory sequence is bound. The nature of these regulatory sequences depends on the host organism. In certain embodiments, the regulatory sequences of the prokaryotes comprise a promoter, a ribosomal binding site and a transcription termination sequence. In other specific embodiments, the regulatory sequences of the eukaryote include a promoter, a transcriptional enhancer sequence and a transcription termination sequence comprising one or multiple recognition sites for a transcription factor. In certain embodiments, a "regulatory sequence" comprises a leader sequence and / or a fusion partner sequence.

The term "polynucleotide" means a single- or double-stranded nucleic acid polymer consisting of at least 10 bases in length. In certain embodiments, nucleotides, including polynucleotides, are ribonucleotides or deoxyribonucleotides or modified forms of nucleotides of either type. Such modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2 ′, 3′-dideoxyribose and phosphorothioate, phosphorodithioate, phosphorosenoate, phosphate Internucleotide binding modifications such as porodiselenoate, phosphoroanilothioate, phosphoroaniladeate and phosphoroamideate. The polynucleotide includes DNA in single stranded and double stranded form.

The term "oligonucleotide" means a polynucleotide comprising 200 bases or more in length. In a preferred embodiment, the oligonucleotides are 10 to 60 bases in length. In a more preferred embodiment, the oligonucleotides are 12, 13, 14, 15, 16, 17, 18, 19 or 20 to 40 bases in length. Oligonucleotides are, for example, single stranded or double stranded to construct a mutant gene. Oligonucleotides of the invention are sense or antisense oligonucleotides.

The term “naturally occurring nucleotide” includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotide" includes nucleotides having a modified or substituted pull or a modified or substituted base. The term "oligonucleotide bond" refers to a bond such as phosphorothioate, phosphorodithioate, phosphorosenoate, phosphorosenoate, phosphoroanilothioate, phosphoroanilideate, phosphoroamideate and the like. It includes. For example, LaPlanche et al. (1986), Nucl. Acids Res. 14: 9081; Stec et al. (1984), J. Am. Chem. Soc. 106: 6077; Stein et al. (1988), Nucl. Acids Res. 16: 3209; Zon et al. (1991), Anti-Cancer Drug Design 6: 539; Zon et al. (1991), Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed.), Oxford University Press, Oxford England; US Patent No. 5,151,510 to Stec et al .; See Uhlmann and Peyman (1990), Chemical Reviews 90: 543. Oligonucleotides of the invention include labels comprising radiolabels, fluorescent labels, heptanes or antigenic labels for detection assays.

The term "vector" refers to any molecule (eg, nucleic acid, plasmid or virus) used to convey coding information to a host cell.

The term “expression vector” or “expression construct” refers to a vector comprising a nucleic acid sequence that is suitable for transforming a host cell and directs and / or regulates the expression of one or more heterologous coding regions that are operably linked to the nucleic acid sequence. it means. Expression constructs include, but are not limited to, sequences that affect or regulate transcription, translation, and RNA splicing of coding regions operably linked to an expression construct, when introns are present.

The term "host cell" refers to a cell that has been or can be transformed with a nucleic acid sequence, thereby expressing a selected gene of interest. The term includes, as long as the selected gene is present, a descendant of the parent cell, whether or not the descendant is morphologically or genetically identical to the parent cell.

The term “transduction” means that the gene is generally transferred from one bacterium to another by phage. "Transduction" also refers to the acquisition and delivery of eukaryotic cell sequences by retroviruses.

The term "transfection" means that the foreign DNA or exogenous DNA is taken up by the cell, and the cell is "transfected" when the exogenous DNA is introduced into the cell membrane. Many transfection techniques are well known in the art and described herein. See, eg, Graham et al. 1973, Virology 52: 456; 2001, Molecular Cloning: A Laboratory Manual, Id. ; 1986, Basic Methods in Molecular Biology, Elsevier; And Chu et al., 1981, Gene 13: 197. Such techniques can be used to introduce one or more exogenous DNA components into suitable host cells.

The term “transformation” means that the genetic properties of a cell are changed, and the cell is transformed when the cell is modified to contain new DNA. For example, a cell is transformed when the cell is genetically modified from its natural state by transfection, transduction or other technique. After transfection or transduction, the transforming DNA can be physically synthesized into the cell's chromosome to be temporarily retained as an episomal component without being recombined or replicated with the cell's DNA or independently replicated as a polamide. . The cell is considered to be "stable transformed" when the transforming DNA is replicated upon cell division.

The term “antigen” refers to a molecule or portion of a molecule that can be used in an animal to produce a molecule that can be bound by an optional binder such as an antibody and additionally an antibody that can bind to the epitope of the antigen. An antigen has one or more epitopes.

The term “epitope” includes determinants, preferably polypeptide determinants, capable of specifically binding to immunoglobulins or T-cell receptors. In certain embodiments, epitope determinants include chemically active surface groups such as amino acids, sugar side chains, phosphoryls or sulfonyls, and in certain embodiments, epitope determinants have three-dimensional structural characteristics and / or specific charge characteristics. Indicates. Epitopes are regions of antigen to which antibodies bind. In certain embodiments, the antibody specifically binds to the antigen when the antibody selectively recognizes the target antigen in a complex mixture of proteins and / or macromolecules. In a preferred embodiment, the dissociation constant is less than or equal to about 10 nM, more preferably if the dissociation constant is less than or equal to about 100 pM, most preferably when the dissociation constant is less than or equal to about 10 pM , The antibody binds specifically to the antigen.

The term “identity” refers to a relationship measured by comparing the sequence of two or more polypeptide molecules or the sequence between two or more nucleic acid molecules. As used herein, "identity" refers to the degree of sequence relationship between a polypeptide or nucleic acid molecule and, as the case may be, determines a match between two or more nucleotides or two or more amino acid sequences. "Identity" is a measure of the same percentage of match between gap arrangements recorded in a particular mathematical model or computer program (ie, "algorithm") that exists at any number and less than or equal to two sequences.

The term "similarity" is used in this field in a related concept that is not in contrast to "identity", but "similarity" also means a measure of relevance, including identical matches and conservative substitution matches. If both polypeptide sequences have, for example, 10/20 identical amino acids and the rest are both non-conservative substitutions, the percentage of identity and similarity are both 50%. In the same embodiment, if there are five or more positions where conservative substitutions are present, the percentage of identity is 50%, but the percentage of similarity is 75% (15/20). Thus, if conservative substitutions are present, the percent similarity between the two polypeptides will be higher than the percent identity between the two polypeptides.

Identity and similarity of related nucleic acids and polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in the literature: Computational Molecular Biology, (Le sk, A.M., ed.), 1988, Oxford University Press, New York; Biocomputing: Informatics and Genome Projects, (Smith, D. W., ed.), 1993, Academic Press, New York; Computer Analysis of Sequence Data, Part 1, (Griffin, A.M. and Griffin, H.G., eds.), 1994, Humana Press, New Jersey; von Heinje, G., Sequence Analysis in Molecular Biology, 1987, Academic Press; Carillo et al. 1988, SIAM J. Applied Math. 48: 1073; And 1998, Biological Sequence Analysis, Cambridge University Press.

Preferred methods for determining identity were designed so that the sequences tested matched one another. Methods of determining identity are described as publicly available computer programs. Preferred computer program methods for determining identity between two sequences are described in the GCG program package including GAP [Devereux et al. 1984, Nucl. Acid. Res. See 12: 387; Genetics Computer Group, obtained from the University of Wisconsin, Madison, Wisconsin, USA], BLASTP, BLASTN and FASTA [Altschul et al., 1990, J. Mol. Biol. 215: 403-410]. The BLASTX program includes National Center for Biotechnology Information (NCBI) and other sources [NCB / NLM / NIH Bethesda, MD 20894 by BLAST Manual, Altschul et al .; 1990, supra, Altschul et al., Supra. The well known Smith Waterman algorithm can also be used to determine identity.

Specific alignment schemes for aligning two amino acid sequences can only match short regions of the two sequences, and even though there is no significant association between the two sequences of the full length, such small regions have very high sequence identity. Thus, in certain embodiments, the alignment method selected (GAP program) spans at least 50 consecutive amino acids of the target polypeptide.

For example, using the computer algorithm GAP (Genetics Computer Group, obtained from the University of Wisconsin, Madison, Wisconsin, USA), two polypeptides to determine percent sequence identity were aligned to optimally match their respective amino acids (algorithm). "Matched length" as determined by). In a particular embodiment, the gap opening penalty (calculated as 3 X average diagonal) as well as the control matrix, such as PAM 250 or BLOSUM 62; "average diagonal" is the average of the diagonals of the control matrix used; Is a score or number defined for each perfect amino acid match by a particular control matrix) and gap elongation penalty (generally 1/10 times the gap opening penalty) can be used in conjunction with the algorithm. In certain embodiments, standard control matrices [Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5: 345-352 on PAM 250 control matrices; Henikoff et al. 1992, Proc. Natl. Acad. Sci USA 89: 10915-10919 may also be used in the algorithm.

In certain embodiments, the parameters for polypeptide sequence comparison include:

Algorithm: Needleman et al. (1970), J. Mol. Biol. 48: 443-453 Reference Control Matrix: Henikoff et al. (1992), BLOSUM 62 Gap Penalty: 12 Gap Elongation Penalty: 4 Threshold of Similarity: 0. The GAP program is effective to perform with these parameters. In certain embodiments, the above mentioned parameters are default parameters for polypeptide comparison (no penalty for terminal gap) using the GAP algorithm.

The term "naturally occurring" as used means the general 20 amino acids. See, eg, Immunology--A Synthesis, 2nd Edition, (E. S. Golub and D. R. Gren, eds.), Sinauer Associates: Sunderland, MA (1991), which is incorporated herein by reference for all purposes.

Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties similar to those of template peptides. Non-peptide compounds of this type are termed "peptide analogs" or "peptide analogs". For example, Fauchere (1986), Adv. Drug Res. 15:29; Veder & Freidinger, 1985, TINS p. 392; And Evans et al. (1987), J. Med. Chem. See 30: 1229. Such compounds are often shaped with the help of computer molecular modeling. Peptide analogs structurally similar to therapeutically useful peptides exhibit similar therapeutic or prophylactic effects. In general, peptidomimetics are structurally similar to typical peptides or polypeptides (ie, peptides or polypeptides with biochemical or pharmacological activity), such as human antibodies, but are well known in the art and may be selected from -CH _2- Out of NH—, —CH ₂ —S—, —CH ₂ —CH ₂ —, —CH═CH— (cis and trans), —COCH ₂ —, —CH (OH) CH ₂ —, and —CH ₂ SO-; Have one or more peptide bonds optionally substituted with a selected bond. Systematic substitution of one or more amino acids of a consensus sequence with the same type of D-amino acid (eg, D-lysine instead of L-lysine) may be used to produce more stable peptides in certain embodiments. have. In addition, methods known in the art (see Rizo & Gierasch, 1992, Ann. Rev. Biochem. 61: 387, incorporated herein by reference for all purposes), for example, to make the peptide cyclic By adding internal cysteine residues that can form intermolecular disulfide bonds, one can generate limited peptides that comprise a co-sequence or substantially identical co-sequence variants.

"Antibody" or "antibody peptide (class)" refers to a complete antibody or a binding fragment thereof that competes with a specific antibody that specifically binds, including chimeric antibodies, humanized antibodies, fully human antibodies, and nonspecific antibodies. In certain embodiments, the binding fragments are made by recombinant DNA technology. In additional embodiments, binding fragments are prepared by enzymatic or chemical cleavage of the complete antibody. Binding fragments include, but are not limited to, Fab, Fab ', F (ab') ₂ , Fv, and single chain antibodies.

The term “heavy chain” includes full length heavy chains and fragments thereof having variable region sequences sufficient to confer specificity for IL-1R1. The full length heavy chain includes the variable region domain, V _H and three constant region domains, C _H 1, C _H 2 and C _H 3. The V _H domain is at the amino-terminus of the polypeptide and the C _H 3 domain is at the carboxy-terminus.

The term “light chain” includes full length light chains and fragments thereof having sufficient variable region sequences to confer specificity for IL-1R1. The full length light chain comprises the variable region domain, V _L and the constant region domain, C _L. Like the heavy chain, the variable region domain of the light chain is at the amino-terminus of the polypeptide.

“Fab fragment” consists of the variable region of one light chain and C _H 1 and one heavy chain. The heavy chain of the Fab molecule cannot form disulfide bonds with other heavy chain molecules.

"Fab 'fragment" includes one light chain and one heavy chain comprising both additional constant regions, C _H 1 and C _H 2, wherein two disulfide bonds between the chains forming the F (ab') ₂ molecule It can be formed between heavy chains.

"F (ab ') ₂ fragment" comprises two heavy chains comprising both the C _H 1 and C _H 2 domains that are part of two light and constant regions, and a disulfide bond between the chains is formed between the two heavy chains .

"Fv region" includes the variable regions of the heavy and light chains but does not include the constant regions.

A “single chain antibody” is an Fv molecule in which heavy and light chain variable regions are linked by a flexible linker to form a single chain polypeptide that forms an antigen-binding region. Single chain antibodies are discussed in detail in International Patent Application Publication Nos. WO 88/01649 and US Pat. Nos. 4,946,778 and 5,260,203, which are incorporated herein by reference for all purposes.

In certain embodiments, it is understood that "bispecific" or "bivalent" antibodies that differ from "multispecific" or "multifunctional" antibodies include binding sites with the same antigenic specificity.

An “bispecific” or “bifunctional” antibody is a hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be prepared in a variety of ways, including but not limited to hybridoma fusion or binding of Fab ′ fragments. See, eg, Songsivilai & Lachmann (1990), Clin. Exp. Immunol. 79: 315-321; Kostelny et al. (1992), J. Immunol. See 148: 1547-1553.

In evaluating antibody binding and specificity of the invention, the amount of receptor to which the excess antibody binds to the counterreceptor is at least about 20%, 40%, 60%, 80%, 85% or more ( When reduced by an ex vivo competitive binding assay, the antibody "substantially inhibits" the binding of the ligand to the receptor.

The term "formulation" means a compound, a mixture of compounds, an extract prepared from a biological macromolecule or a biological substance.

The term "label" or "labeled" refers to, for example, a radiolabeled amino acid or a target avidin (eg, a fluorescent marker, chemiluminescent marker or enzymatic activity, preferably detectable by optical or colorimetric methods, for example). Streptavidin comprising a detectable marker) to bind the detectable marker to the polypeptide of the biotin component detectable. In certain embodiments, the label is also therapeutic. Various methods of labeling polypeptides and glycoproteins are known in the art and can be advantageously used with the methods described herein. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (eg, ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ^{99 m} Tc, ¹¹¹ In, ¹²⁵ I , ¹³¹ I), fluorescent labels (eg, fluorescent isothiocyanates (FITC), rhodamine or lanthanide), enzymatic labels (eg, horseradish peroxidase, β-galactosidase, By luciferase, alkaline phosphatase), chemiluminescent labels, heptane labels such as biotinyl groups and secondary receptors (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope labels) Already determined polypeptide epitope recognized. In certain embodiments, the label is bound to spacer arms of various lengths (eg, (CH ₂ ) _n when n <about 20) to reduce potential steric hindrance.

The term "biological sample" includes, but is not limited to, any amount of material obtained from living or living organisms. Such organisms include, but are not limited to, humans, mice, monkeys, rats, rabbits, and other animals. The substance may be blood, serum, urine, cells, organs, tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue, chondrocytes, synovial macrophage, endothelial cells, vascular tissue (especially inflammation). Vascular tissue) and skin). The terms "pharmaceutical agent" and "drug" refer to compounds or compositions which, when properly administered to a patient, can induce the desired therapeutic effect.

The term "patient" includes human and animal subjects.

Except as otherwise required herein, the singular terms include the plural and the plural terms include the singular.

amino acid

Twenty naturally occurring amino acids and their abbreviations are commonly used. See Immunology--A Sytnhesis, 2nd Edition, (E. S. Golub and D. R. Gren, eds.), Sinauer Associates: Sunderland, MA (1991), which is incorporated herein by reference for all purposes. The stereoisomers of twenty conventional amino acids (eg, D-amino acids), non-natural amino acids such as α-, α-unsubstituted amino acids, N-alkyl amino acids and other non-common amino acids are also polypeptides of the invention. Is a suitable ingredient for. Examples of unusual amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N, N, N-trimethyllysine, ε-N-acetyllysine, O-phosphoseline, N-acetylserine , N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, σ-N-methylarginine and other similar amino acids and imino acids (eg, 4-hydroxyproline). In the polypeptide notation used herein, according to the conventions used in the standard, the left direction is the amino terminal direction and the right direction is the carboxy terminal direction.

Except when similarly specified otherwise, the left end of the single-stranded polynucleotide sequence is the 5 'end; The left direction of the double-stranded polynucleotide sequence is the 5 'end. The direction from 5 'to 3' to which the generated RNA transcript is added is called a transcription direction; The sequence region from the 5 'to 5' end of an RNA transcript in a DNA strand having the same sequence as the RNA transcript is called an "upstream sequence"; The sequence region from the 3 'to 3' end of an RNA transcript in a DNA strand having the same sequence as the RNA transcript is referred to as the "downstream sequence".

Naturally occurring amino acid residues can be classified into the following classes according to conventional side chain properties:

1) hydrophobic: norleucine (Nor or Nle), Met, Ala, Val, Leu, Ile; 2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; 3) acidic: Asp, Glu; 4) basic: His, Lys, Arg; 5) residues affecting chain origin: Gly, Pro; And 6) aromatic: Trp, Tyr, Phe.

Conservative amino acid substitutions include the replacement of a member of one of the classes with another member of the same class. Conservative amino acid substitutions include non-naturally occurring amino acid residues, which generally include those by chemical peptide synthesis rather than those synthesized in the biological world. Such amino acids include peptidomide and other reverse phase amino acid components.

Non-conservative substitutions include replacement of one member of the class with a member of another class. Substituted residues can be introduced, for example, into regions of human antibodies that are homologous to non-human antibodies or into non-homologous regions of a molecule.

In substitutions according to certain embodiments, hydrotherapy values of amino acids are contemplated. Each amino acid is determined hydrotherapy values based on its hydrophobicity and charge characteristics. This figure is as follows: Isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cystine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartic acid salts (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The importance of amino acid hydrotherapy values that confer interactive biological function on proteins is understood in the art (see, eg, Kyte et al. 1982, J. Mol. Biol. 157: 105-131). It is known that certain amino acids can be substituted with other amino acids having similar hydrotherapy values and have similar biological activities. In certain embodiments substituting based on hydrotherapy values includes substitution of amino acids having hydrotherapy values within ± 2. In certain embodiments, those having numerical values within ± 1 are included and in certain embodiments, those within ± 0.5 are also included.

It is understood in the art that analogous amino acids are effectively substituted on the basis of hydrophilicity, in particular the biologically functional proteins or peptides resulting from such substitutions are used in immunological embodiments as described herein. In certain embodiments, the very local mean hydrophilicity of a protein regulated by the hydrophilicity of adjacent amino acids relates to its immunogenicity and antigenicity, ie the biological properties of the protein.

Hydrophilicity values of amino acid residues are shown below: arginine (+3.0); Lysine (+3.0); Asparagine salts (+3.0 ± 1); Glutamate (+3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); And tryptophan (-3.4). Substitutions based on similar hydrophilicity values include, in certain embodiments, substitutions of amino acids with hydrophilicity values within ± 2 and substitutions of amino acids with hydrophilicity values within ± 1 and amino acids within ± 0.5. Epitopes can also be identified from primary amino acid sequences based on hydrophilicity values. Such regions are referred to as "epitope core regions".

General amino acid substitutions are shown in Table 1.

Those skilled in the art can determine suitable variants of the polypeptides as described herein using well known techniques. In certain embodiments, one of ordinary skill in the art can identify appropriate portions of the molecule that are substituted with target regions that do not disrupt the activity but do not significantly affect the activity. In certain embodiments, one skilled in the art can identify residues and portions of molecules that are conserved among analogous polypeptides. In further embodiments, even regions that have a significant effect on biological activity or structure can be made conservative amino acid substitutions without destroying biological activity or adversely affecting polypeptide structure.

In addition, those skilled in the art can review structure-acting studies that identify residues of similar polypeptides that are important for activity or structure. Through this comparison, the skilled person can predict the importance of amino acid residues in a protein corresponding to amino acid residues that are important for the activity or structure of the analogous protein. Those skilled in the art can substitute chemically similar amino acids for amino acid residues predicted to be important amino acid residues.

Those skilled in the art can also analyze amino acid sequences and three-dimensional structures regarding the structure of analogous polypeptides. Those skilled in the art can use this information to predict the arrangement of amino acid residues of an antibody with respect to the three-dimensional structure of the antibody. In certain embodiments, such residues relate to important interactions with other molecules, so those skilled in the art can choose not to cause radical changes to amino acid residues expected at the surface of the protein. In addition, those skilled in the art can make experimental variants in which a single amino acid is substituted at each preferred amino acid residue. This variant can then be screened using activity assays known to those skilled in the art. Such variants can be used to collect information about suitable variants. For example, if a change in specific amino acid residues results in loss of activity, undesirably reduced or inadequate, then variants with this variation are preferred. That is, one of ordinary skill in the art can readily determine the amino acids so that additional variations do not occur alone or in combination with other mutations, based on information gathered from general experiments.

There have been many scientific presentations predicting secondary structure. Moult, 1996, Curr. Op. in Biotech. 7: 422-427; Chou et al. 1974, Biochemistry 13: 222-245; Chou et al. 1974, Biochemistry 113: 211-222; Chou et al. 1978, Adv. Enzymol. Relat. Areas Mol. Biol. 47: 45-148; Chou et al. 1979, Ann. Rev. Biochem. 47: 251-276; And Chou et al. 1979, Biophys. See J. 26: 367-384. In addition, computer programs that are helpful in predicting secondary structure are generally available. One way of estimating secondary structure is based on homology modeling. For example, two polypeptides or proteins with at least 30% sequence identity or at least 40% similarity often have similar structural topologies. Recent developments in protein structure databases (PDBs) have increased the predictability of secondary structures, including the number of possible folds in polypeptide or protein structures. Holm et al. 1999, Nucl. Acid. Res. See 27: 244-247. There is a limited number of folds in a particular polypeptide or protein and it is suggested that once the critical number of structures is established, the prediction of the structure will be accurate (Brenner et al. 1997, Curr. Op. Struct. Biol. 7: 369). -376).

An additional method of predicting secondary structure is "threading" (see Jones, 1997, Curr. Opin. Struct. Biol. 7: 377-87; 1996, Structure, 4: 15-19 to Sippl et al.) , "Profile Analysis" (Bowie et al. 1991, Science 253: 164-170; Gribskov et al. 1990, Meth. Enzym. 183: 146-159; Gribskov et al. 1987, Proc. Nat. Acad. Sci. 84: 4355- 4358] and "affinity associations" (Holm 1999, supra; and Brenner's 1997, supra).

In certain embodiments, antibody variants include glycosylation variants, wherein the number and / or type of glycosylation sites is mutated as compared to the amino acid sequence of the morphopeptide. In certain embodiments, protein variants comprise more or less N-linked glycosylation sites than natural proteins. The N-linked glycosylation site is characterized by the following sequence: Asn-X-Ser or Asn-X-Thr, where the amino acid residues represented by X are all amino acid residues except proline. Substitution of the amino acid residues that produce such sequences provides a novel site to which N-linked carbohydrate chains can be added. Optionally, the N-linked carbohydrate chains present were removed with substitutions to remove these sequences. In addition, one or more N-linked glycosylation sites (generally naturally occurring sites) are removed to provide rearrangement of the N-linked carbohydrate chains resulting in one or more new N-binding sites. Additional preferred antibody variants include cysteine variants in which one or more cysteine residues are deleted from the parent amino acid sequence or substituted with a corresponding amino acid (eg, serine). Cysteine variants are useful when the antibody is folded back into a biologically active conformation after separating insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than natural proteins and generally have enough cysteine residues to minimize interactions due to unpaired cysteine.

In certain embodiments, amino acid substitutions reduce (1) susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) change binding affinity to form protein complexes, and (4) Change binding affinity and / or (5) confer or modify physicochemical or functional properties on such polypeptides. In certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made of naturally occurring sequences (in certain embodiments, part of a polypeptide outer domain that forms an intermolecular bond). In preferred embodiments, conservative amino acid substitutions generally do not significantly alter the structural characteristics of the sequence (e.g., replacement amino acids do not destroy the helix that occurs in the sequence or other types of features characterized by the sequence). Does not collapse the secondary structure). Examples of polypeptide secondary structures and tertiary structures recognized in the art include Proteins, Structures and Molecular Principles, (Creighton, ed.), 1984, W. H. Freeman and Company, New York, each of which is incorporated herein by reference; Introduction to Protein Structure (C. Branden and J. Tooze, eds.), 1991, Garland Publishing, New York. N.Y. ; And Thornton et al. (1991), Nature 354: 105.

Preparation of Antibodies

Naturally occurring antibody structural units generally include tetramers. Each tetramer generally comprises two identical pairs of polypeptide chains, each pair having one full length "light" chain (typically having a molecular weight of about 25 kDa) and one full length "heavy" chain (Generally having a molecular weight of about 50-70 kDa). The amino-terminal portion of each chain generally comprises a variable region consisting of about 100 to 110 or more amino acids, which generally recognizes the antigen. The carboxy-terminal portion of each chain is usually specified as a constant region that can function as an effector. Human light chains are generally classified into κ and λ light chains. Heavy chains are generally classified into μ, σ, γ, α and ε, each of which is defined as antibody isotypes such as IgM, IgD, IgG, IgA and IgE. IgG has a variety of subclasses, including but not limited to IgG1, IgG2, IgG3 and IgG4. IgM has subclasses, including but not limited to IgM1 and IgM2. IgA is similarly classified into subclasses including, but not limited to, IgA1 and IgA2. In general, in the full length light and heavy chains, the "J" region consisting of about 12 or more amino acids joins the constant and variable regions with a heavy chain comprising a "D" region consisting of about 10 or more amino acids. . For example, Fundamental Immunology, Ch. 7, 2 ^nd ed., (Paul, W., ed.), 1989, Raven Press, NY, which is incorporated herein by reference. The combination of variable regions of each light / heavy chain pair generally forms an antigen-binding site.

The variable regions of each heavy and light chain represent the same general structure, including four relatively conserved outer structural regions (FRs) that bind three hypervariable regions, commonly referred to as complementary determining regions or CDRs. The CDRs of each of the two chains of each pair are bound to the outer structural region, the arrangement of which is bindable to a particular epitope. From the N-terminus to the C-terminus, the light and heavy chain variable regions generally comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The amino acid sequence for each domain is generally described by Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, National Institutes of Health, Bethesda, Md.), Chothia & Lesk, 1987, J. Mol. Biol. 196: 901-917, or Chothia et al., 1989, Nature 342: 878-883).

The development of monoclonal antibodies has made them useful and interesting as pharmaceutical preparations. Monoclonal antibodies are produced using any method of producing antibody molecules by continuously culturing cell lines. Examples of suitable methods for preparing monoclonal antibodies are the hybridoma method described in Kohler et al. (1975, Nature 256: 495-497) and the human B-cell hybridoma method [Kozbor, 1984, J. Immunol]. . 133: 3001; And 1987, Monoclonal Antibody Production Techniques and Applications, (Marcel Dekker, Inc., New York), pp. Brodeur et al. 51-63].

Monoclonal antibodies can be modified for use as therapeutic agents. One example is that some of the heavy and / or light chains are identical or homologous to the corresponding sequence of an antibody derived from a particular species or an antibody belonging to a particular antibody class or subclass, while the rest of the chain is an antibody or other antibody derived from another species. A "chimeric" antibody that is identical or homologous to the sequence of the antibody belonging to the class or subclass. As long as the fragments exhibit desirable biological activity, other examples are fragments of such antibodies. See, for example, US Pat. No. 4,816,567; And Morrison et al. (1985), Proc. Natl. Acad. Sci. See USA 81: 6851-6855. A related developing antibody is a “CDR-transplanted” antibody, which antibody comprises one or more complementary determining regions (CDRs) obtained from a particular species or one or more complementary determining regions belonging to a particular antibody class or subclass. While the rest of the antibody chain is identical or homologous to the corresponding sequence of an antibody derived from another species or an antibody of another antibody class or subclass.

Another developing antibody is a "humanized" antibody. Methods of humanizing non-human antibodies are well known in the art (see, eg, US Pat. Nos. 5,585,089 and 5,693,762). In general, humanized antibodies are prepared from non-human animals and then generally modify certain amino acid residues of the non-antigen recognition portion of the antibody to be homologous to the corresponding residues of the corresponding isotype human antibody. Humanization is a method described in the art, for example, by substituting at least a portion of a variable region of a rodent for a corresponding region of a human antibody (Jones et al. 1996, Nature 321: 522-525; Riechmann et al. 1988, Nature 332: 323-327; 1988, Science 239: 1534-1536 to Verhoeyen et al.

Generating human antibodies (“complete human antibodies”) without exposing the human body to antigens is a newer and more promising method. Using a transformed mouse capable of producing human antibodies in the absence of endogenous mouse immunoglobulins, the antibody is produced by immunization with an antigen (generally having at least six contiguous amino acids) optionally bound to a carrier. can do. See, eg, Jakobovits et al. 1993, Proc. Natl. Acad. Sci. USA 90: 2551-2555; 1993, Nature 362: 255-258 by Jakobovits et al .; And 1993, Year in Immunol, Bruggermann et al. See 7:33. In one example of this method, a transformed animal is generated by disabling its endogenous mouse immunoglobulin locus encoding its mouse immunoglobulin heavy and light chains and inserting a locus encoding human heavy and light chain proteins into its genome. can do. Partially modified animals with modifications that are not completely complementary are hybridized to obtain animals with all desired immune system modifications. When administering an immunogen, the transformed animal produces an antibody that is immunospecific against an antigen having human amino acid sequence rather than a mouse comprising a variable region. See, eg, PCT Publication Nos. WO 96/33735 and WO 94/02602, which are incorporated by reference. Additional methods are described in US Pat. No. 5,545,807, PCT Publication Nos. WO 91/10741, WO 90/04036, and EP 546063B1 and EP 546073A1, which are incorporated by reference. Human antibodies can be prepared by expressing recombinant DNA of a host cell as described herein or by expressing in hybridoma cells.

Fully human antibodies can also be prepared with phage-display libraries (described in 1991, J. Mol. Biol. 227: 381 by Hoogenboom et al. And 1991, J. Mol. Biol. 222: 581 by Marks et al.). . This method is similar to the method of expressing an antibody on the surface of a fibrous bacteriophage for immunoselection and then binding to a selected antigen to select phage. This method is described in PCT Publication No. WO 99/10494, which is incorporated by reference, and is used to isolate high active and functional functional antibodies against MPL- and msk-receptors using such methods. Is also described.

Nucleotide sequences encoding such antibodies have been determined and chimeric antibodies, CDR-grafted antibodies, humanized antibodies and fully human antibodies can also be prepared by recombinant methods. Nucleic acids encoding antibodies by materials and methods generally known in the art are introduced into the host cell and then expressed.

The present invention provides one or more fully human monoclonal antibodies that bind to human IL-1R1. Preferably, the antibody binds to the third domain of IL-1R1. In a preferred embodiment, the invention provides amino acid sequences comprising sequences corresponding to heavy and light chain immunoglobulin molecules, in particular their variable regions, and nucleotide sequences encoding them. In a preferred embodiment, the present invention provides sequences corresponding to complementary determining regions (CDRs), in particular CDR1 to CDR3. In a further preferred embodiment, the present invention provides hybridoma cell lines expressing immunoglobulin molecules produced in cell lines and monoclonal antibodies, most preferably purified human monoclonal antibodies that bind to human IL-1R1. .

The ability to clone and reconstruct the million-sized human locus of artificial yeast chromosomes (YACs) and introduce artificial yeast chromosomes into mouse germ cells not only creates a useful model for human disease, but also maps very large or natural maps. It provides an advantageous way to identify the functional components of the prepared loci. Moreover, the use of techniques to replace the mouse lobe with human equivalents provide insight into the expression and regulation of human gene products during development, interaction with other systems, and their relevance in disease development and progression.

An important practical method of this strategy is the "humanization" of the mouse humoral immune system. Introduction of human immunoglobulin (Ig) loci into mice in which endogenous Ig genes are inactivated provides an opportunity to study the mechanisms underlying the programmed expression and synthesis of antibodies as well as the role of antibodies in B-cell development. do. This method also provides a circle for producing fully human monoclonal antibodies (MAbs), in particular a circle for use as a therapeutic agent. It is anticipated that fully human antibodies will enhance efficacy and stability when administered antibodies therapeutically by minimizing inherent immunogenic and allergic responses to mice or mouse-induced Mabs. Fully human antibodies can be used to treat chronic and recurrent human diseases such as osteoarthritis, rheumatoid arthritis and other inflammatory symptoms and to treat diseases that require repeated administration of antibodies.

Those skilled in the art can genetically engineer mouse strain with large fragments of the human Ig locus to prevent the production of mouse antibodies so that mice produce human antibodies without producing mouse antibodies. Large human Ig fragments can not only moderate antibody production and expression, but also preserve a variety of large gene diversity. By exploiting mice and their lack of immunological resistance to human proteins for antibody diversification and selection, human antibodies reproduced from engineered mouse strains have a high activity of binding to all antigens of interest, including human antigens. Calculate the antibody. Hybridoma technology can be used to generate and select antigen-specific human MAbs with desirable specificity.

In certain embodiments, one of skill in the art can use human variable regions in mice and constant regions of humans and other species together to produce chimeric antibodies. Antibodies of the invention can be produced by immunizing an animal having a full-length IL-1R1, a soluble form of IL-1R1, or a fragment thereof. See, eg, International Patent Application Publication No. WO 93/12227.

CDRs of the light and heavy chain variable regions of the anti-IL-1R1 antibodies of the invention can be implanted into outer structural regions (FRs) from the same or different species. In certain embodiments, CDRs of the light and heavy chain variable regions of the anti-IL-1R1 antibody can be transplanted into common human FRs. To generate consensus human FRs, FRs from several human heavy or light chain amino acid sequences were listed to identify consensus amino acid sequences. The anti-IL-1R1 antibody heavy or light chain FRs can be replaced with FRs of other heavy or light chains. Generally, trace amino acids are not substituted in the FRs of the heavy and light chains of the anti-IL-1R1 antibody, but the remaining FR amino acids may be substituted. Trace amino acids are specific amino acids at positions not normally found in FRs. The implanted variable region in the anti-IL-1R1 antibody of the invention can be used with the constant region and other constant regions of the anti-IL-1R1 antibody. Optionally, the implanted variable region is part of a single chain Fv antibody. CDR transplantation is described, for example, in US Pat. Nos. 6,180,370, 5,693,762, 5,693,761, 5,585,089, and 5,530,101, which are incorporated herein by reference.

In certain embodiments, the invention provides an anti-IL1-R1 antibody comprising: a human heavy chain CDR 1 region having the amino acid sequence of SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63; Human heavy chain CDR 2 region having the amino acid sequence of SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66; And / or the human heavy chain CDR 3 region having the amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69.

In another embodiment, the invention provides an anti-IL1-R1 antibody comprising: a human light chain CDR 1 region having the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 71; Human heavy chain CDR 2 region having the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 73; And / or the human heavy chain CDR 3 region having the amino acid sequence of SEQ ID NO: 74 or SEQ ID NO: 75.

Antibodies of the invention are preferably prepared using transgenic mice that have substantial portions of the locus that produce human antibodies inserted into cells that produce antibodies of the mouse, and that have been further engineered to produce no antibodies of endogenous mice. Such mice have the ability to produce human immunoglobulin molecules and antibodies and produce or do not produce substantially reduced amounts of immunoglobulin molecules and antibodies. The techniques used to obtain these results are described in patents, applications, and references described herein. In a preferred embodiment, the methods used by those skilled in the art are described in International Patent Application Publication No. WO 98/24893, incorporated herein by reference. See Mendez et al., 1997, Nature Genetics 15 : 146-156, incorporated herein by reference.

The monoclonal antibodies (MAbs) of the invention can be produced by a variety of techniques including general monoclonal antibody methodology (see, eg, standard somatic cell hybridization technique of Kohler and Milstein, 1975, Nature 256 : 495). have. It is preferred to use somatic hybridization methods, but generally other techniques for producing monoclonal antibodies (e.g., transformation of B-lymphocyte virus or tumor gene transformation) can be used.

In a preferred embodiment, endogenous μ And a mouse referred to as a "HuMab" mouse containing the human immunoglobulin gene minilocus, which is an unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, with target mutations that inactivate the κ chain locus. Can be used to produce human monoclonal antibodies directed against IL-1R1. See Lonberg et al., 1994, Nature 368: 856-859. Thus, mice will exhibit reduced expression of mouse IgM or κ and, in the immunization response, perform class switch and somatic mutations on human heavy and light chain transgenes introduced to produce human IgG κ monoclonal antibodies with high binding capacity. It was. Lonberg et al., Supra; Lonberg and Huszar, 1995, Intern. Rev. Immunol. 13 : 65-93; Harding and Lonberg, 1995, Ann. NY Acad. Sci. 764 : 536-546. The preparation of HuMab mice is described in Taylor et al., 1992, Nucleic Acids Res. 20 : 6287-6295; Chen et al., 1993, International Immunology 5 : 647-656; Tuaillon et al., 1994, J. Immunol. 152 : 2912-2920; Lonberg et al., 1994, Nature 368 : 856-859; Lonberg, 1994, Handbook of Exp. Pharmacology 113 : 49-101; Taylor et al., 1994, International Immunology 6 : 579-591; Lonberg & Huszar, 1995, Intern. Rev. Immunol. 13 : 65-93; Harding & Lonberg, 1995, Ann. NY Acad. Sci 764 : 536-546; Fishwild et al., 1996, Nature Biotechnology 14 : 845-851. See also US Pat. No. 5,545,806, which is further incorporated herein by reference; 5,569,825; 5,569,825; 5,625,126; 5,625,126; 5,633,425; 5,633,425; 5,789,650; 5,789,650; 5,877,397; 5,877,397; 5,661,016; 5,661,016; 5,814,318; 5,814,318; 5,874,299; 5,874,299; And 5,770,429; In US Pat. No. 5,545,807 to Surani et al., As well as all references to Lonberg and Kay et al .; International Patent Application Publication No. WO 93/1227 published June 24, 1993; WO 92/22646 published December 23, 1992; And WO 92/03918, published March 19, 1992. Alternatively, the HCo7 and HCo12 transgenic mouse strains described in the Examples below can be used to produce human anti-IL-1R1 antibodies.

Conveniently, complete human monoclonal antibodies specific for IL-1R1 were prepared as follows. Transgenic mice containing human immunoglobulin genes were immunized with the desired IL-1R1-associated antigen. Lymph cells (such as B-cells) were obtained from mice in which antibodies were expressed. These cells recovered to generate immortalized hybridoma cell lines are fused with bone marrow-type cell lines and screened and screened for such hybridoma cell lines to identify hybridoma cell lines that produce antibodies specific for the desired antigen. It was. In certain embodiments, production of hybridoma cell lines that produce antibodies specific for IL-1R1 is provided.

In a preferred embodiment, antibodies of the invention were produced by hybridoma cell lines. In this embodiment, the antibodies of the invention bound IL-1R1 with dissociation constants (K _d ) of approximately 4 pM to 100 pM. In certain embodiments of the invention, the antibody binds to IL-1R1 with a K _d of less than about 20 pM. In another embodiment, the antibody of the invention binds to the third domain of IL-1R1. The nucleotide sequence and amino acid sequence of the third domain of human and rat IL1-R1 are shown in FIG. 17.

In a preferred embodiment, the antibody of the invention is an IgG1, IgG2 or IgG4 isotype, with the IgG2 isotype being most preferred. In a preferred embodiment of the invention, the antibody comprises human κ light chain and human IgGl, IgG2 or IgG4 heavy chain. In certain embodiments, the variable region of the antibody is associated with a constant region other than the constant region of an IgG1, IgG2 or IgG4 isotype. In certain embodiments, antibodies of the invention were cloned for expression in mammalian cells.

In certain embodiments, conservative amino acid substitutions (and modifications consistent with coding nucleotides) of the heavy and light chains of the anti-IL-1R1 antibody exhibit functional and chemical properties similar to those of the heavy and light chains of the anti-IL-1R1 antibody. It is possible to produce an anti-IL-1R1 antibody. In contrast, substantial modifications in the functional and / or chemical properties of the anti-IL-1R1 antibody may be indicated by substitutions selected from the amino acid sequences of the heavy and light chains, wherein the amino acid sequences of the heavy and light chains are (a) substituted In maintaining the structure of the molecular skeleton in the region (eg, sheet conformation or helix conformation), (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chains These effects are very different.

For example, “conservative amino acid substitutions” include substitution of natural amino acid residues with non-natural residues that have little or no effect on the polarity or charge of the amino acid residues at that position. In addition, certain natural residues in the polypeptide may also be substituted with alanine, which is "alanine scanning mutagenesis" [Wells, 1991, Methods Enzymol. 202 : 390 (ed JJ Langone), Academic Press, London.

If such substitutions are desired, the required amino acid substitutions (regardless of conservative or non-conservative substitutions) can be determined by one skilled in the art. In certain embodiments, amino acid substitutions can be used to identify important residues of the anti-IL-1R1 antibodies described herein or to increase or decrease the affinity of the anti-IL-1R1 antibodies.

In alternative embodiments, the antibodies of the invention may be expressed in cell lines other than hybridoma cell lines. In such embodiments, sequences encoding particular antibodies can be used for transformation of appropriate mammalian host cells. In accordance with this embodiment, polynucleotide introduction into a host cell, including, for example, polynucleotide packaging in a virus (or into a viral vector) and host cell transduction using a virus (or a vector) is employed. Transformation can be carried out using well known methods or transfection methods well known in the art. Such methods are illustrated in US Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455, which are incorporated herein by reference. In general, the transformation method used depends on the host to be transformed. Methods of introducing heterologous polynucleotides into mammalian cells are well known in the art and include, but are not limited to: dextran-mediated transfection, potassium phosphate precipitation, polybrene-mediated transfection, protoplast fusion , Electroporation, polynucleotide encapsulation in liposomes and direct microinjection of DNA into the nucleus.

According to certain embodiments of the methods of the invention, nucleic acid molecules encoding the amino acid sequences of the heavy chain constant region, heavy chain variable region, light chain constant region or light chain variable region of the IL-1R1 antibody of the invention may be used in standard ligation techniques. ) Into the appropriate expression vector. In a preferred embodiment, the IL-1R1 heavy or light chain constant region is added to the C-terminus of the appropriate variable region and bound to the expression vector. Vectors were selected to be functional in the particular host cell generally used (ie, the vector may be compatible with host cell machinery such as amplification and / or expression of a gene). For a review of expression vectors, see Goeddel (ed.), 1990, Meth. Enzymol. Vol. 185, Academic Press. See N.Y.

In general, expression vectors used in all host cells will include sequences for plasmid maintenance and sequences for cloning and expression of exogenous nucleotide sequences. Such sequences, referred to collectively as "flanking sequences" in certain embodiments, will generally include one or more nucleotide sequences, such as: promoter, one or more enhancer sequences, origin of replication, transcription A complete intron sequence comprising a termination sequence, a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a liposome binding site, a polyadenylation sequence, a polylinker region for inserting a nucleic acid encoding a polypeptide to be expressed And selectable marker elements. Each of these sequences is discussed below.

Optionally, the vector comprises a "marker" -coding sequence (ie, an oligonucleotide molecule located at the 5 'or 3' end of the IL-1R1 polypeptide coding sequence); The oligonucleotide sequence encodes polyHis (such as hexaHis) or other marker such as myc present in FLAG, HA (hemagglutinin influenza virus), or commercially available antibodies. This marker is generally fused to the polypeptide depending on the expression of the polypeptide and can be used as an affinity purification or as a means of detecting the IL-1R1 antibody from the host cell. Affinity purification can be performed using, for example, column chromatography using an antibody against a label as an affinity matrix. Optionally, the label can be removed from the purified IL-1R1 polypeptide by various means, such as using specific peptidase for cleavage.

The flanking sequences may be homologous (ie, from the same species and / or same lineage as the host cell), heterologous (ie, from a species or lineage other than the host cell), hybrid (ie, flanking from more than one source) Combination of sequences], synthetic or natural. If the flanking sequence is functional and can be activated in host cell tissue, the source of the flanking sequence can be obtained from all prokaryotes or eukaryotes, all vertebrate or invertebrates, or all plants.

Flanking sequences useful in the vectors of the invention can be obtained by a variety of methods well known in the art. In general, flanking sequences useful herein can be identified by mapping and / or restriction endonuclease cleavage and thus can be isolated from appropriate tissue using appropriate restriction endonucleases. In some cases, the complete nucleotide sequence of the flanking sequence is well known. Here, flanking sequences can be synthesized using the methods described herein for nucleic acid synthesis or cloning.

Genome library using polymerase chain reaction (PCR) and / or using appropriate probes such as oligonucleotides and / or flanking sequence fragments from the same species or other species, if only all or part of the flanking sequence is known Can be obtained by screening. If the flanking sequence is unknown, a fragment of DNA comprising the flanking sequence can be isolated from, for example, a large piece of DNA comprising a coding sequence or other gene or genes. Cleavage with restriction endonuclease to produce appropriate DNA fragments followed by isolation using agarose gel purification, Qiagen ^® column chromatography (obtained from Chatsworth, Calif.) Or other methods well known to those skilled in the art. . The selection of appropriate enzymes for use in this method will be apparent to those skilled in the art.

The origin of replication is generally part of a commercially available prokaryotic expression vector, the origin of which aids in the amplification of the vector in host cells. If the selected vector does not contain the origin of replication, it can be chemically synthesized based on known sequences and can be bound to the vector. For example, the origin of replication from plasmid pBR322 (obtained from New England Biolabs, Beverly, Mass.) Is suitable as the starting point for most Gram-negative bacteria and various viral starting points [eg, SV40, Folio Hemp, adenovirus, vesicular stomatitus virus (VSV) or papilloma virus such as HPV or BPV] are useful as cloning vectors in mammalian cells. In general, the starting point of the replication moiety does not require a mammalian expression vector (eg, the SV40 starting point is often used because it includes the viral early promoter).

The transcription terminal sequence is generally located at terminal 3 'of the polypeptide coding region and serves to terminate transcription. In general, the transcription termination sequence in prokaryotic cells is a poly-T sequence followed by a G-C rich fragment. Such sequences can be easily cloned from a library or even commercially available portions of a vector, while can be readily synthesized using nucleic acid synthesis methods such as those described herein.

Selectable marker genes encode proteins essential for host cell growth and survival in selective culture media. Common selection marker genes encode the following proteins: (a) proteins that are resistant to antibiotics or other toxins (eg, ampicillin, tetracycline or kanamycin) in prokaryotic host cells; (b) proteins that compensate for nutrient deficiencies in cells; Or (c) a protein that supplies important nutrients that are not available from complete or specific media. Preferred selectable markers are kanamycin resistance gene, ampicillin resistance gene and tetracycline resistance gene. Neomycin resistance genes can also be selected and used in both prokaryotic and eukaryotic host cells.

Other selectable genes can be used to amplify the gene to be expressed. Amplification is a process in which genes that require more production of proteins important for cell survival or growth are typically repeated in series in the chromosome to continue to occur in recombinant cells. Examples of suitable selectable markers in mammalian cells include dihydrofolate reductase (DHFR) and promoter deficient thymidine kinase. Mammalian cell transformants were placed under selection pressure, where the transformants were only adapted and survived by selectable genes present in the vector. The selection pressure cultures the transformed cells under conditions that continuously increase the concentration of the selection agent in the medium, thereby amplifying both DNA encoding other genes, such as IL-1R1 polypeptides, including selectable genes and vectors. This is by derivation. As a result, increased quantities of polypeptides, such as IL-1R1 polypeptides, were synthesized from amplified DNA.

Ribosome-binding sites are generally essential for initiation of translation of mRNA and are characterized by shine-dalgarno sequences (prokaryotes) or Kozak sequences (eukaryotes). This element is generally located 3 'to the promoter and 5' to the coding sequence of the polypeptide to be expressed.

In some cases, glycosylation is required in the eukaryotic host cell expression system, which can manipulate various pre- or prosequences to increase glycosylation or yield. For example, a peptidase cleavage site of a particular single peptide that may affect glycosylation can be altered or presequences added. The final protein product may have one or more additional amino acids that are likely to be expressed that are not entirely removed at one position (the first amino acid of the mature protein). For example, the final protein product may have one or two amino acid residues at the peptidase cleavage site attached to the amino-terminus. Optionally, in the case of cleavage using enzymes in these regions within a mature polypeptide, the use of several enzyme cleavage sites results in truncated forms of the desired polypeptide.

Expression and cloning vectors of the invention generally include a promoter that is recognizable by the host organism and capable of binding to a molecule encoding an anti-IL-1R1 antibody. The promoter is an untranscribed sequence (typically within about 100 bp to 1000 bp) located upstream of the start codon of the structural gene (ie, 5 ') that regulates the transcription of the structural gene. Promoters are usually included in one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under the control of inducible promoters in response to changes in culture conditions, such as the presence or absence of nutrients, or changes in temperature. In contrast, the constitutive promoter initiates continuous gene product production, with little or no regulation in gene expression. Many promoters that are recognized by a variety of potential host cells are well known. Suitable promoters may be bound to DNA encoding the heavy or light chain comprising an anti-IL-1R1 antibody of the invention by removing the promoter from the original DNA by restriction digestion and inserting the required promoter sequence into the vector.

Suitable promoters for use in yeast hosts are also well known in the art. Yeast enhancers can be used advantageously with yeast promoters. Suitable promoters for use in mammalian host cells are well known and include, but are not limited to, those obtained from the genome of the following viruses: polyoma virus, chickenpox virus, adenovirus (such as adenovirus 2), bovine Papilloma virus, chicken sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus and most preferably Simian virus 40 (SV 40). Other suitable mammalian promoters include heterologous mammalian promoters (eg, heat shock promoters and actin promoters).

Additional promoters include, but are not limited to: the SV 40 early promoter region (Bernoist and Chambon, 1981, Nature 290 : 304-10); CMV promoter; Promoters involved in the 3 ′ long terminal repeat of the Raus sarcoma virus (Yamamoto et al., 1980, Cell 22 : 787-97); Herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78 : 1444-45); Regulatory sequences of metal thionine genes (Brinster et al., 1982, Nature 296 : 39-42); Prokaryotic expression vectors such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75 : 3727-31); Or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80 : 21-25). It also has tissue specificity and has the following animal transcriptional regulatory regions that can be used in transgenic animals: elastase I gene regulatory regions active in pancreatic acinar cells (Swift et al., 1984, Cell 38 : 639-46; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50 : 399-409 (1986); MacDonald, 1987, Hepatology 7 : 425-515); Insulin gene regulatory regions active in pancreatic beta cells (Hanahan, 1985, Nature 315 : 115-22); Immunoglobulin gene regulatory regions active in lymphoid cells (Grosschedl et al., 1984, Cell 38 : 647-58; Adames et al., 1985, Nature 318 : 533-38; Alexander et al., 1987, Mol. Cell. Biol. 7 : 1436-44); Tumor virus regulatory regions of mouse breasts active in testicular cells, breast cells, lymphoid cells and mast cells (Leder et al., 1986, Cell 45 : 485-95); Albumin gene regulatory regions active in the liver (Pinkert et al., 1987, Genes and Devel. 1 : 268-76); Α-fetoprotein gene regulatory regions active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5 : 1639-48; Hammer et al., 1987, Science 235 : 53-58); Α1-antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devel 1 : 161-71); Β-globin gene regulatory region active in myeloid cells (Mogram et al., 1985, Nature 315 : 338-40; Kollias et al., 1986, Cell 46 : 89-94); Myelin basic protein gene regulatory regions active in oligodendrocytes in the brain (Readhead et al., 1987, Cell 48 : 703-12); Myosin light chain-2 gene regulatory region active in skeletal muscle (Sani, 1985, Nature 314 : 283-86); And gonadotropin free gene regulatory regions active in the hypothalamus (Mason et al., 1986, Science 234 : 1372-78).

Enhancer sequences can be inserted into the vector to increase the transcription of the DNA encoding the light or heavy chain comprising the anti-IL-1R1 antibody of the invention by higher eukaryotes. Enhancers are cis-acting elements of DNA about 10-300 bp long and act on the promoter to increase transcription. Enhancers are relatively direction-independent and position-independent. Enhancers are found at 5 'and 3' of the transcription unit. Several enhancer sequences available from mammalian genes (ie, globin, elastase, albumin, α-feto-protein and insulin) are well known. However, in general, enhancers from viruses are used. SV 40 enhancers, cytomegalovirus early promoter enhancers, polyoma enhancers and adenovirus enhancers, which are well known in the art, are preferred facilitating factors for activation of promoters in eukaryotes. Enhancers can be spliced into a vector at position 5 'or 3' of the nucleic acid molecule, which is generally located at the 5 'site of the promoter.

Expression vectors of the invention can be constructed from initiation vectors, such as commercially available vectors. Such vectors may or may not contain all of the desired flanking sequences. If one or more flanking sequences described herein are not present in the vector, they can be obtained separately and bound to the vector. The method used for obtaining each flanking sequence is well known to those skilled in the art.

After constructing the vector and inserting the light chain containing the anti-IL-1R1 antibody or the nucleic acid molecule encoding the light chain and the heavy chain and the heavy chain into the appropriate site of the vector, the completed vector is amplified and / or a suitable host cell for polypeptide expression. Can be inserted into Transformation of expression vectors for anti-IL-1R1 antibodies into selected host cells can be carried out by well known methods such as: transfection, injection, calcium phosphate co-precipitation, electroporation, microinjection Lipofection, DEAE-dextran-mediated transfection or other known technique. The method chosen depends on the type of host to be used. Such and other suitable methods are well known to those skilled in the art and are described, for example, in Sambrook et al., Supra.

When cultured under appropriate conditions, the host cell synthesized an anti-IL-1R1 antibody, which can be collected from the culture medium if the host cell secretes the antibody into the medium, or the host cell producing it if not secreted. It can be obtained directly from. Selection of an appropriate host cell will depend on several factors, such as the level of expression required, polypeptide modifications desired or essential for activity (eg glycosylation or phosphorylation) and ease of folding into biologically active molecules.

Mammalian cell lines as hosts for expression are well known in the art and include, but are not limited to, many immortalized cell lines available from the American Type Culture Collection (ATCC), including: Chinese Hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg Hep G2) and many other cell lines. In certain embodiments, the cell line can be selected by whether the cell line has high expression levels and produces antibodies with constitutive IL-1R1 binding properties. In another embodiment, cell lines from B cell lines (eg, mouse myeloma cell lines NS0 and SP2 / 0) that are unable to produce antibodies but have the ability to make and produce heterologous antibodies can be selected.

Antibodies of the invention are useful for screening IL-1R1 in biological samples and for identifying cells or tissues that produce IL-1R1 protein. The antibodies that bind IL-1R1 and prevent interaction with other binding compounds have therapeutic uses to modulate IL-1 mediated diseases. In a preferred embodiment, the antibody of IL-1R1 can block IL-1R1 binding to IL-1β or IL-1α, which can lead to disruption of the IL-1 signal transduction cascade.

Antibodies of the invention that specifically bind to IL-1R1 may be useful for the treatment of IL-1 mediated diseases described below. The antibodies are useful for binding assays to detect IL-1R1 binding and have the ability to inhibit IL-1R1 from complex formation with IL-1β and IL-1R accessory protein (IL-1RAcP) or IL-1α and IL-1RacP. Can have.

In certain embodiments, the present invention provides a method for treating a disease associated with an IL-1 mediated inflammatory response or an IL-1 mediated immunomodulatory response. The method of the invention includes a method of administering an anti-IL1R1 antibody of the invention to a patient suffering from an inflammatory or immunomodulatory disease mediated by IL-1. As used herein, the terms "disease", "disease", "medical symptoms" or "abnormal symptoms" may be used interchangeably with the term "medical disorder".

In certain embodiments, the methods of the present invention comprise administering to a patient an anti-IL-1R1 antibody of the invention that prevents binding of IL-1 to its cell surface receptor (IL-1R1).

In order to treat a disease characterized by abnormal or excessive expression of IL-1 or by abnormal or excessive signaling of IL-1, the IL of the invention to induce sustained improvement in at least one indicator reflecting the severity of the disease A sufficient amount of the molecule comprising the -1R type I antibody was administered to the patient for a sufficient time. If the patient improves in at least two cases divided between 1 and 4 weeks, the improvement is considered to be “continuous”. The degree of improvement is determined based on the signs or symptoms and a questionnaire, such as a questionnaire about the quality of life, may be used for the administered patient.

Various indicators reflecting the extent of the disease of the patient can be evaluated to determine if the amount and time of treatment is sufficient. Baseline values for selected indicators are determined by the patient's examination prior to administering the first dose of antibody. Preferably, the baseline test is made within about 60 days of the first dose administration. If the IL-1R antibody is administered to treat acute symptoms such as trauma injury (knee injury, stroke, head injury, etc.), the first dose is given as soon as possible after the injury or event occurs.

Improvement is induced by repeated administration of the antibody until improvement above the baseline of the selected indicator is indicated. In chronic symptomatic treatment, the degree of improvement can be obtained by repeated administration of the drug for a period of at least one month or more (eg, one month, two months or three months or longer or indefinitely). A period of one to six weeks or even one administration is sufficient to treat acute symptoms.

Although the extent of a patient's disease may appear to be enhanced according to one or more indicators after treatment, treatment may continue indefinitely at the same level or may continue treatment with a reduced dose or frequency. Once treatment is reduced or stopped, treatment should resume at its original level if symptoms later reappear.

An effective route of administration can be used to administer the antibody therapeutically. Antibodies can be injected via intraarticular, intravenous, intramuscular, intralesional, intraperitoneal, intracranial, inhalation or subcutaneous routes by injection or continuous infusion. For example, lung disease may use intranasal and inhalation methods. Other suitable means of administration include sustained release of the insert, aerosol inhalation, eye drops, oral formulations (pills, syrups, glycosides or chewing gums) and topical formulations (lotions, gels, sprays, ointments) or other suitable techniques. . Administration by inhalation is particularly beneficial when treating diseases associated with lung disease.

In one embodiment of the invention, the anti-IL-1R1 antibody of the invention may be administered once a month. In another embodiment, the antibody is administered once every two weeks or once a week to treat the various diseases described herein. In another embodiment, the antibody is administered at least twice per week and in another embodiment, at least once daily. Adult patients are patients over 18 years old. When injected, the effective amount of an adult dose is in the range of 1-200 mg / m 2 or 1-40 mg / m 2 or about 5-25 mg / m 2. Alternatively, administration may be in the range of 2-400 mg / dose, 2-100 mg / dose or about 10-80 mg / dose. If administered more than once per week, typical dosage ranges are less than or equal to the previously described dosage ranges. In one embodiment of the invention, the various indicators described below are treatable by administration of an acceptable agent comprising an 80-100 mg / dose or optionally 80 mg / dose of an IL-1 receptor antibody. Doses were administered repeatedly. When using a route of administration other than injection, the dosage can be appropriately adjusted according to standard medical practice. For example, if the route of administration is inhalation, the dosage can be administered 1 to 7 times a week at a dosage ranging from 10 mg / dose to 50 mg / dose.

In a preferred embodiment, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of an antibody of the invention in combination with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. Preferably, the acceptable formulation material is nontoxic at the dosages and concentrations used for the recipient. In a preferred embodiment, there is provided a pharmaceutical composition comprising a therapeutically effective amount of an anti-IL-1R1 antibody.

In certain embodiments, the pharmaceutical composition modifies, maintains or preserves, for example, the pH, osmolarity, viscosity, purity, color, isotonicity, flavor, sterilization, stability, dissolution or release rate, adsorption or permeation of the composition. Formulation materials for In such embodiments, suitable formulation materials include, but are not limited to: amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); Antimicrobial agents; Antioxidants (eg, ascorbic acid, sodium sulfate or sodium hydrogen sulfide); Buffer solutions (eg, borate, bicarbonate, tris-HCl, citrate, phosphate or other organic acids); Volume control agents (eg mannitol or glycine); Chelating agents [eg, ethylenediamine tetraacetic acid (EDTA)]; Complexing agents (eg, caffeine, polyvinylpyrrolidone, β-cyclodextine or hydroxypropyl-β-cyclodextrin); Filler; Monosaccharides; Disaccharides; And other carbohydrates (eg, glucose, mannose, or dextrins); Proteins (eg, serum albumin, gelatin or immunoglobulins); Pigments, antiseptics and diluents; Emulsifiers; Hydrophilic polymers (eg, polyvinylpyrrolidone); Low molecular weight polypeptides; Salt-forming counterions (eg sodium); Preservatives (eg, benzalkonium chloride, benzoic acid, salicylic acid, chimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); Solvents (eg glycerin, propylene glycol or polyethylene glycol); Sugar alcohols (eg, mannitol or sorbitol); Suspending agents; Surfactants or wetting agents (eg, polysorbates such as pluronic, PEG, sorbitan esters, polysorbate 20, polysorbate 80, tritone, tromethamine, lecithin, cholesterol and tiloxapal); Stability enhancers (eg sucrose or sorbitol); Enteric enhancers (eg, alkali metal halogen compounds, preferably sodium chloride, potassium chloride, mannitol sorbitol); Transport excipients; Diluent; Prosthetics; And / or pharmaceutical adjuvants. See Remington's Pharmaceutical Sciences, 18 ^th Edition, (AR Gennaro, ed.) 1990, obtained from Mac Publishing Company, which is incorporated herein by reference.

In certain embodiments, the optimal pharmaceutical composition will be determined by one of ordinary skill in the art, for example, according to the intended route of administration, mode of transport and dosage required. See, eg, Remington's Pharmaceutical Sciences, supra. In certain embodiments, the composition affects the rate of in vivo clearance, rate of in vivo release, stability, and physical state of the antibodies of the invention.

In certain embodiments, the primary excipient or carrier in the pharmaceutical composition is aqueous or non-aqueous in nature. For example, a suitable excipient or carrier may be water for injection, physiological saline or artificial cerebrospinal fluid, which is generally capable of supplementing other substances in the composition for parenteral administration. Neutral buffered saline or physiological saline mixed with serum albumin is an additional example of an excipient. In a preferred embodiment, the pharmaceutical composition comprises Tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which further comprises sorbitol or a suitable substituent thereof. In certain embodiments of the invention, the anti-IL-1R1 antibody composition is mixed with a selection composition having the desired purity and an optimal formulation formulation (see Remington's Pharmaceutical Sciences, cited above), or a lyophilized cake or Prepared for storage in the form of an aqueous solution. Moreover, in certain embodiments, the anti-IL-1R1 antibody product is formulated as a lyophilisate using a suitable prosthetic such as sucrose.

The pharmaceutical compositions of the invention can be selected for parenteral administration. The composition was selected for transport through the digestive tract, such as oral, or for inhalation. Preparation of such pharmaceutically acceptable compositions is within the skill of the art.

The formulation component is preferably present at an acceptable concentration at the site of administration. In certain embodiments, buffers were used to maintain the composition at physiological pH or slightly lower pH, generally from about pH 5 to about pH 8.

If intended for parenteral administration, the therapeutic composition used in the present invention provides a form of a pyrogen-free parenterally acceptable aqueous solution comprising the required anti-IL-1R1 antibody dissolved in a pharmaceutically acceptable excipient. do. Particularly suitable excipients for parenteral injection are sterile distilled water, which formulates the anti-IL-1R1 antibody as a sterile isotonic solution, which is suitably preserved. In certain embodiments, the formulations are injectable microparticles, erodible particles, polymeric compounds (polylactic and polyglycolic acid), beads, or which provide controlled release and sustained release products that can be transported via depot injection. Formulations of the required molecules with agents such as liposomes. In certain embodiments, hyaluronic acid can also be used, which has the effect of increasing the duration in circulation. In certain embodiments, an implantable drug transporter can be used to inject the desired antibody molecule.

The pharmaceutical compositions of the present invention may be formulated for inhalation. In such embodiments, the anti-IL-1R1 antibody may be formulated as a dry powder for inhalation. In a preferred embodiment, the anti-IL-1R1 antibody inhalation solution can also be formulated with a propellant for aerosol transport. In certain embodiments, the solution may be sprayed. Thus, methods for administering and formulating lungs are further described in International Patent Publication No. WO 94/20069, which describes the lung transport of chemically modified proteins, which is incorporated herein by reference.

The formulations can also be administered orally. The anti-IL-1R1 antibody administered in this manner may be formulated with or without a carrier generally used in the preparation of solid pharmaceutical formulations such as tablets and capsules. In certain embodiments, the capsule may be designed to release the effective portion of the formulation at a gastrointestinal tract location where the bioavailability is maximized and pre-systemic degradation is minimized. Additional agents may be included to facilitate uptake of anti-IL-1R1 antibodies. Diluents, flavors, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be used.

The pharmaceutical compositions of the present invention preferably provide for the inclusion of an effective amount of one or most anti-IL-1R1 antibodies in admixture with non-toxic excipients suitable for the manufacture of tablets. By dissolving the tablets in sterile water or other suitable excipients, solutions may be prepared in unit pharmaceutical formulations. Suitable excipients include, but are not limited to: inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; Or binders such as starch, gelatin or acacia; Or lubricants such as magnesium stearate, stearic acid or talc.

Additional pharmaceutical compositions comprising a formulation comprising an anti-IL-1R1 antibody in a sustained release transport formulation will be apparent to those skilled in the art. Formulation techniques for liposome carriers, bio-corrosive microparticles or other various sustained release vehicles such as porous beads and depot injections are also well known to those skilled in the art. See International Patent Publication No. WO93 / 15722, incorporated herein by reference, which describes the sustained release of porous polymeric microparticles for the transport of pharmaceutical compositions. Sustained-release preparations may include semipermeable polymer matrices in the form of shaped drugs (eg, films or microcapsules). Sustained release matrixes may include: polyesters, hydrogels, polylactides (described in US Pat. No. 3,773,919 and European Patent Application No. EP 058481), L-glutamic acid, and γ ethyl- Copolymers of L-glutamate (Sidman et al., 1983, Biopolymers 22 : 545-556), poly (2-hydroxyethyl-methylacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15 : 167-277 and Langer, 1982, Chem. Tech. 12 : 98-105), ethylene vinyl acetate (from Langer et al.) Or poly-D (-)-3-hydroxybutyric acid (European Patent Application Publication No. EP 133,988). Sustained release compositions may also include liposomes, which may be prepared by various methods known in the art. See, eg, Eppstein et al. 1985, Proc. Natl. Acad. Sci, USA 82 : 3688-3692; European Patent Application Publication No. EP 036,676; See EP 088,046 and EP 143,949.

Pharmaceutical compositions used for administration in vivo are generally provided as sterile preparations. It can be sterilized by filtration through sterile filtration membranes. When the composition was lyophilized, it was sterilized using this method before or after lyophilization and reconstitution. The composition for parenteral administration was stored in lyophilized form or as a solvent. Parenteral compositions are generally stored in containers with sterile access ports, such as vials or intravenous solution bags with stoppers that can be inserted into a hypodermic needle.

Once the pharmaceutical composition has been formulated, it is stored in sterile vials in the form of solvents, suspensions, gels, emulsions, solids or dehydrogenated or lyophilized powders. Such formulations are stored in an easy-to-use form or in a form that requires reconstitution (eg, lyophilized) prior to administration.

The invention also provides kits for providing single-dose dosage units. Kits of the invention each comprise both a primary container containing dried protein and a secondary container containing an aqueous formulation. In certain embodiments of the present invention, the kits provide single and multi-threaded prefield cyclers (eg, liquid syringes and lyosyringes).

The therapeutically effective amount of a pharmaceutical composition containing a therapeutically used anti-IL-1R1 antibody depends, for example, on the therapeutic situation and the subject. Therefore, those skilled in the art will appreciate that dosage levels suitable for treatment may be at least partially indicative of the use of transport molecules, anti-IL-1R1 antibodies, route and size of administration (weight, body surface area or organ size) and / or symptoms of the patient ( Age and overall health). In certain embodiments, the clinician may titrate the dosage and alter the route of administration to obtain the optimal therapeutic effect. Typical dosages range from about 0.1 μg / kg up to about 100 mg / kg or more, depending on the factors mentioned above. In a preferred embodiment, the dosage range is from 0.1 μg / kg to about 100 mg / kg; More preferably 1 μg / kg to about 100 mg / kg; Even more preferably from 5 μg / kg to about 100 mg / kg.

The number of administrations will depend on the pharmacokinetic parameters of the particular anti-IL-1R1 antibody of the formulation used. In general, the clinician has administered the composition until the dosage achieves the desired effect. Therefore, the compositions were administered by a single dose or by two or more doses (with or without the same amount of the desired molecule) or by continuous infusion via an insertion device or catheter over time. Moreover, refining at a suitable dosage is an area of common work typically made by a person skilled in the art and done by a person skilled in the art. Appropriate doses were identified using appropriate dose-response data.

The route of administration of the pharmaceutical composition is according to known methods, including, for example: oral; Injection by intravenous, intraperitoneal, intracranial (brain parenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional routes; Sustained release system; Or injection using an insertion device. In certain embodiments, the composition may be administered by injection or infusion or infusion device continuously.

The composition can be administered topically through the insertion of a membrane, sponge or other suitable material in which the required molecules are absorbed or encapsulated. In certain embodiments, when using an insertion device, the device is inserted into an appropriate tissue or organ and the required molecules are transported by diffusion, sustained-release concentrates or continuous administration.

It would also be desirable to use the anti-IL-1R1 antibody pharmaceutical compositions of the invention ex vivo. In this example, cells, tissues or organs removed from the patient were transplanted back to the patient after exposure to the anti-IL-1R1 antibody pharmaceutical composition.

In particular, anti-IL-1R1 antibodies can be transplanted and transported with specific cells genetically engineered for polypeptide expression and secretion using the methods described herein. In certain embodiments, such cells are animal or human cells and may be autologous, heterologous, or heterologous. In certain embodiments, the cells can be immortalized. In another embodiment, to reduce the chance of an immune response, encapsulation can be avoided to avoid infiltration of tissue surrounding the cells. In a further embodiment, the encapsulated material is generally a biocompatible, semi-permeable polymeric enclosure or membrane for releasing the protein product but this is disruption of cells by other harmful elements from the patient's immune system or surrounding tissues. Can be prevented.

In certain embodiments, the invention further comprises concurrently administering the anti-IL-1R1 antibody or pharmaceutical composition of the invention in combination with one or more other drugs administered to the same patient, wherein each drug is suitable for It was administered according to the curing method. This includes pretreatment, concurrent treatment, continuous treatment and replacement regimens. Examples of such drugs include, but are not limited to: antiviral drugs, antibiotics, analgesics, corticosteroids, antagonists of inflammatory cytokines, disease-modifying anti-rheumatic drugs (DMARDs) and non-steroidal anti- -Inflammatory drugs.

In another embodiment, an anti-IL-1R1 antibody or pharmaceutical composition of the invention comprises other cytokines comprising an antagonist (eg, RANKL, TGFβ, IFNγ, IL-6 or IL-8 and TNF, in particular TNFα). It can be administered in combination with a cain inhibitor. When mixed with IL-6, the antibodies of the invention can be used to prevent and treat recurrences of seizures induced by GABA _A receptor antagonism, seizures associated with episodes of EEG seizures, and motility marginal seizures that occur during epileptic conditions. . When mixed with an IFN _γ inhibitor, the antibodies of the invention are useful for treating idiopathic pulmonary fibrosis and cystic fibrosis. Mixtures of IL-1 receptor antibodies and RANKL inhibitors (eg, RANKL antibodies) are useful for bone destruction in a variety of cases, including but not limited to: various rheumatic diseases, osteoporosis, multiple myeloma or bone degeneration Bone destruction associated with destructive malignancies, or anti-tumor treatments or prostheses that wear to prevent bone metastasis, or debris or periodontal tissue wear. In addition, the antibodies of the present invention can be administered in combination with the following IL-17 inhibitors: dissolved forms of the IL-17 receptor (IL-17R: Fc) or IL-17 antibodies or IL-17R antibodies, IL- 18 binding protein, lysed form of IL-18 receptor and IL-18 antibody, antibody against IL-18 receptor or antibody against CD30-ligand or CD4.

The present invention makes additional TNF inhibitors, preferably a TNFR: Fc, and was mixed with (ENBREL ^®), including how to use the -IL1R1 wherein the antibody or pharmaceutical composition of the present invention for the treatment of diseases described herein, as described above Saito Mixtures of caine or cytokine inhibitors are effective agents in combination therapy. For example, in accordance with the present invention, the combination treatment method can be used to treat rheumatoid arthritis, seizures, asthma, psoriasis and the like.

Symptoms that are effectively treated by the anti-IL-1R1 antibodies or pharmaceutical compositions described herein include the following lung diseases, and include antibodies and IL-4 inhibitors and / or IL-13 inhibitors of IL-1R (e.g. For example, IL-4R antibodies that inhibit IL-13 and IL-4 activity) can be used in combination: asthma, chronic obstructive pulmonary disease, alveolar proteinosis, bleomyosine-induced pulmonary disease and fibrosis, radio-induced pulmonary fibrosis , Cystic fibrosis, collagen accumulation in the lungs and ARDS. The antibodies herein and the pharmaceutical compositions of the invention are also useful for the treatment of the following diseases: bronchial-lung dysplasia (BPD); Chronic obstructive pulmonary disease (eg emphysema and chronic bronchitis), chronic fibrosis lung disease in infants. In addition, the compounds, compositions, and combination treatments of the present invention have been used to treat occupational lung diseases including asbestosis, coal miner pneumoconiosis, silicosis, or similar symptoms associated with prolonged microparticle exposure. In another aspect of the invention, the compounds, compositions and combination treatments are useful for treating the following diseases: pulmonary fibrosis, including bronchioliterans stroma pneumonia, idiopathic pulmonary fibrosis and radiation-induced pulmonary fibrosis; Pulmonary sarcoidosis; And allergies including allergic rhinitis, contact dermatitis, atopic dermatitis and asthma.

Such compounds are useful for treating patients suffering from a variety of skin diseases, including but not limited to: hives and vulgaris including herpes dermatitis (Turing disease), atopic dermatitis, contact dermatitis, chronic idiopathic urticaria Autoimmune blebs disease, including awards and bullous hereditary rewards. Other diseases treatable with a mixture of IL-1R antibodies and IL-4 and / or IL-13 inhibitors include: myesthenia gravis, sarcoidosis including pulmonary sarcoidosis, scleroderma, reactivity Arthritis, excess IgE syndrome, multiple sclerosis and idiopathic hypereosinophilic syndrome. The mixture was used as an adjuvant of medication and allergy immunotherapy to treat allergic reactions.

The IL-1 receptor antibodies and pharmaceutical compositions described herein are useful for the treatment of protozoa diseases including malaria and schistosomiasis and are known to be inferior to crystalline erythema; Bacterial or viral meningitis; Tuberculosis, including pulmonary tuberculosis; It is useful for treating secondary pneumonia of bacterial or viral infections, including influenza infections and infectious cytomegaly.

Cardiovascular disease and injury is treatable and / or preventable in combination with only one of the pharmaceutical compositions or anti-IL1-R1 antibodies or with other cytokine inhibitors. Treatable cardiovascular diseases include: aortic aneurysm of the abdomen, acute coronary syndrome, aortic aneurysm including arteritis; Vascular occlusion, including cerebral artery occlusion; Complications of coronary bypass surgery; Focal anemia / reperfusion injury; Heart disease, including atherosclerotic heart disease; Myocarditis, including chronic autoimmune myocarditis and viral myocarditis; Heart failure, including chronic heart failure, congestive heart failure, cachexia of heart failure; Myocardial infarction; Recurrent stenosis and / or atherosclerosis after cardiac surgery or surgery of carotid angioplasty; Asymptomatic myocardial ischemia; Pump dysfunction of the left ventricle, complications after implantation of assist device of the left ventricle; Raynaud 's phenomenon; Thrombophlebitis; Vasculitis, including Kawasaki vasculitis; Venous obstructive disease, giant cell arteritis, Wegener's granulomatosis; Mental confusion and Cardinal-Henoch Zaban after cardiopulmonary bypass surgery.

In certain embodiments, the anti-IL-1R1 antibodies and pharmaceutical compositions of the invention can also be used to treat chronic pain symptoms such as chronic pelvic pain, including chronic prostatitis / pelvic pain syndrome and postherpetic pain.

Endocrine diseases including childhood onset diabetes (including autoimmune diabetes and insulin-dependent type diabetes) and mature onset diabetes (including non-insulin-dependent diabetes and obesity-mediated diabetes) include anti-IL of the present invention. It can also be treated with -1R1 antibody or pharmaceutical composition. Such diseases include diabetic retinopathy, renal transplant rejection in diabetic patients, secondary symptoms associated with obesity-mediated insulin resistance, and renal failure associated with proteinurea and hypertension. Other endocrine diseases can be treated with these compounds and endocrine diseases include polycystic ovary disease, X-related adrenal protein dystrophy, thyroid dysfunction and thyroiditis including Hashimoto's thyroiditis (ie, autoimmune thyroiditis), normal thyroid patient syndrome Thyroid cell dysfunction.

The anti-IL-1R1 antibodies or pharmaceutical compositions of the invention alone or in admixture with other therapeutic mixtures are treatable or preventable of symptoms of the gastrointestinal system. These symptoms include abdominal cavity, Crohn's disease; Ulcerative colitis; Idiopathic gastric palsy; Pancreatitis including chronic pancreatitis, acute pancreatitis; Inflammatory bowel disease and ulcers including gastric ulcer and duodenal ulcer.

Diseases of the genitourinary system are also treatable or preventable with the anti-IL-1R1 antibodies or pharmaceutical compositions described herein. Such diseases include glomerulonephritis, including autoimmune glomerulonephritis, glomerulonephritis resulting from exposure to toxin or glomerulonephritis secondary to hemolytic streptococci or other hepatitis factors. Symptoms treatable with the compounds, compositions and therapeutic mixtures of the present invention are also clinical complications (eg, renal failure, anemia and thick myocardium), including uremia syndrome, environmental toxins, drugs or other causes associated with uremia syndrome. Symptoms). Inflammatory complications of the gallbladder wall leading to changes in adsorption function are treatable or preventable with the antibodies of the invention. These complications include gallstones (gallstones) and biliary biliary stones (biliary stones), and cholelithiasis and biliary biliary stones. Treatable symptoms in the compounds, compositions, and combination treatments of the invention include complications of hemodialysis; Prostate symptoms, including benign prostatic hyperplasia, non-bacterial prostatitis and chronic prostatitis; And complications of hemodialysis.

Also provided herein are methods of using the anti-IL-1R1 antibodies, compositions, and combination treatments of the present invention for treating a variety of hematological and tumor disorders. For example, the anti-IL-1R1 antibody described above or in combination with cytokine inhibitors or other active agents can be used to treat various forms of cancer, including: acute myeloid leukemia, chronic myeloid Leukemia, carcinoma of the Epstein-Barr-positive-viral nasopharyngeal pharynx, glioblastoma, colon, stomach, prostate and renal cell carcinoma, cervical cancer, ovarian cancer and cancer-related cachexia, fatigue, weakness, cachexia and atrial syndrome of hypercalcemia Lung cancer comprising (SCLC and NSCLC). Carcinomas such as solid tumors and adenocarcinomas (eg, breast cancer) and squamous cell carcinomas, including sarcomas and osteosarcomas, are also treatable. Additionally treatable cancers include esophageal cancer, gastric cancer, gallbladder carcinoma and leukemias including acute myeloid leukemia, chronic myeloid leukemia, myeloid leukemia, chronic or acute lymphoblastic leukemia and hairy cell leukemia. Other malignancies with invasive metastatic potential, including multiple myeloma, can be treated with compounds, compositions and combination treatments.

In addition, the anti-IL-1R1 antibodies of the present invention can be used to treat the following diseases: chronic idiopathic neutropenia, anemia, including anemia of chronic disease, and disorders of blood, including aconomy aplastic anemia Aplastic anemia; Idiopathic thrombocytopenic purpura (ITP); Thrombotic thrombocytopenic purpura, myelodysplastic syndromes, refractory anemia of annulocytic hairballs, refractory anemia in surplus blast cells, refractory anemia including refractory anemia in surplus blast cells of transformation); Myelofibrosis / myeloid metaplasia; And sickle cell vascular obstruction.

Various lymphocyte proliferative disorders are also treatable with the anti-IL-1R1 antibodies of the invention, including: autoimmune lymphoid proliferative syndrome (ALPS), chronic lymphocytic leukemia, hairy cell leukemia, chronic lymphatic Leukemia, Peripheral T-Cell Lymphoma, Small Lymphoid, Mantle Cell Lymphoma, Follicle Lymphoma, Burkitt's Lymphoma, Epstein-Barr Positive-Virus T Cell Lymphoma, Histoblastic Lymphoma, Hodgkin's Disease, Disseminated Attack Lymphoma , Acute lymphocytic leukocytes, Tγ lymphocyte proliferative disease, B-cell lymphoma of the skin, T-cell lymphoma of the skin (ie, Mycosis Honggoides) and Sedentary syndrome.

Hereditary symptoms such as Gaucher disease, Huntington's disease, linear IgA disease and muscular dystrophy can be treated with the antibodies of the invention.

Other symptoms that can be treated or prevented by the IL-1 receptor antibody or pharmaceutical composition of the invention include diseases from brain or spinal cord injury, including subdural hematoma following brain injury. In connection with such treatment, the compositions and mixtures herein are suitable for the prevention of cranial neurological damage and for the prevention and treatment of cervical headaches. The compositions and mixtures described are further suitable for treating neurological side effects associated with brain irradiation.

The anti-IL-1R1 antibodies and pharmaceutical compositions of the present invention may also be used for acute alcoholic hepatitis, acute drug-induced hepatitis or viral hepatitis, hepatitis A, hepatitis B and C, sclerotic cholangitis, and hepatic vascular epithelium and eggs. It is useful for treating symptoms of the liver, such as hepatitis, including inflammation of the liver caused by missing causes.

Hearing loss related disorders and disorders associated with abnormal IL-1 expression can be treated with the anti-IL-1R1 antibodies or pharmaceutical compositions of the invention. Such disorders include cochlear-related hearing loss (ie, autoimmune hearing loss) as a result of the autoimmune process. Also treatable or preventable symptoms with an anti-IL-1R1 antibody or pharmaceutical composition of the present invention are Menier syndrome and pearloma and otitis media associated with hearing loss.

Non-arthritis disorders of the bones and joints can also be treated with the antibodies described herein. These disorders include osteoclast disorders that cause bone loss, and osteoclast disorders include, but are not limited to: Osteoporosis, including postmenopausal osteoporosis, tooth agitation or teeth caused by osteoarthritis, fasciitis Loss, loosening of prosthesis after joint replacement (generally related to immune response to abrasion of tissue fragments). The latter symptom is also called "orthopedic implant osteolysis." Other symptoms treatable with the compounds, compositions, and combination treatments of the present invention are temporal mandibular joint dysfunction (TMJ).

Anti-IL-1R1 antibodies or pharmaceutical compositions of the invention may also be used in adult and combustible rheumatoid arthritis; Scleroderma; Systemic lupus erythematosus; Gout; Osteoarthritis; Rheumatoid multiple myopathy; Serum tests including ankylosing spondylitis can be used to treat seronegative spondylarthropathy and rheumatic diseases including lighter disease, psoriatic arthritis and chronic Lyme arthritis. Antibodies of the invention are also useful for treating inflammation of the veterinary muscle and other muscles, including dermatitis, inclusion body myositis, polymyositis and lymphangioleimyomatosis.

Other uses of the antibodies and pharmaceutical compositions of the invention include Alzheimer's disease, secondary reactive hereditosis; Down syndrome; And treatment and / or prevention of primary hereditary and secondary hereditary disorders characterized by a variety of symptoms including dialysis-associated genetics. In addition, diseases treatable with the antibodies or pharmaceutical compositions of the invention include hereditary cycles including familial Mediterranean fever, hyperimmunoglobulin D and periodic fever syndrome, and TNF-receptor associated periodic syndrome (TRAPS). It is a fever syndrome.

In another embodiment, the antibodies or pharmaceutical compositions of the invention can also be used to treat disorders related to skin or mucous membranes. Such disorders include polar cytopathic disease, including follicular disease, follicular keratosis and vulgaris. Additional skin diseases that can be treated with the antibodies of the invention include: acne, rosacea, alopecia areata, aphthous stomatitis, bullous swelling, laceration, eczema, polymorphic erythema and bullous polymorphic erythema (Stevens-). Erythema including Johnson syndrome), immune skin disease, lichen planus, linear IgA bullous disease (chronic bullous skin disease in childhood), decreased skin elasticity, mucosal surface ulcers including gastric ulcer, neutrophil dermatitis (Sweet syndrome), Dermatitis, pore red nasal mucus, psoriasis, necrotic pyoderma, multicardiac myocytosis and toxic epidermal detachment. Other skin related symptoms treatable with the treatments and combination therapies of the invention include herpes dermatitis.

Additional disorders that may be treated with the antibodies or pharmaceutical compositions of the present invention include macrohost graft disease and other transplants, including parenchymal organ transplants such as heart, liver, skin, kidneys, lungs (removing lung transplant airways) or bone marrow transplants. Include.

Ocular disorders are also treatable or preventable with the anti-IL-1R1 antibodies or pharmaceutical compositions of the invention, which include inflammatory eye diseases including ruptured retinal detachment and inflammatory eye diseases related to smoking and macular degeneration.

Antibodies or pharmaceutical compositions of the invention as described herein are useful for treating disorders affecting the female reproductive system. Examples of such disorders include, but are not limited to: multiple implant failures / infertility; Fetal loss syndrome or IV embryo loss (natural heritage); Preeclampsia pregnancy or eclampsia; Endometriosis, chronic cervicitis and early delivery.

In addition, the antibodies or pharmaceutical compositions of the present invention are useful for treating and / or preventing sciatica, aging symptoms, severe drug action (eg, IL-2 toxicity or bleomycin-induced pulmonary disease and fibrosis). Or to inhibit transfusion of allogeneic red blood cells or other inflammatory reactions before, during or after cardiac surgery, or to treat traumatic injury of limbs or joints, such as traumatic knee injury. Various other medical disorders treatable with an anti-IL-1R1 antibody or pharmaceutical composition of the present invention include various autoimmune disorders or diseases related to genetic deficiency, including X chromosome associated psychoanalysis, as well as multiple sclerosis; Bethset syndrome; Sjogren's syndrome; Autoimmune hemolytic anemia; β-thalassemia; Proximal lateral sclerosis (Lou Gehrig's disease); Parkinson's disease; And hay salts occurring for unknown reasons.

In addition, anti-IL-1R1 antibodies or pharmaceutical compositions of the invention are useful for treating central nervous system damage, including the effects of neurotoxic neurotransmitters released during stimulation of inflammation in the central nervous system (CNS) It is useful for inhibiting and preventing the development of glial scars. With regard to the treatment of epilepsy and seizures, it reduces the number and severity of seizures, the severity of the detrimental effects of seizures, and reduces neuronal loss, neuronal degeneration and neuroglial hyperplasia associated with seizures.

Additional uses of the antibodies or pharmaceutical compositions of the invention include, but are not limited to, treating the following diseases: severe illness polyneuropathy and myopathy (CIPNM) acute multiple neuropathy; Anorexia nervosa; Bell 's palsy; Chronic fatigue syndrome; Infectious dementia including Creutzfeldt-Jakob disease; Demyelinating neuropathy; Merlang-Barré syndrome; Spinal disc disease; Gulf War Syndrome; Chronic inflammatory demyelinating polyneuropathy, myasthenia gravis; Asymptomatic cerebral ischemia; Sleep disorders including sleep attacks and sleep apneas; Chronic neuronal degeneration; And seizures, including cerebral ischemic disease. Still further uses of the antibodies of the invention include: anorexia and / or anorexia symptoms, peritonitis, endotoxins and sepsis 쇽, acute infections such as granulomatous formation, heat stroke, Chug-Strauss syndrome, tuberculosis and leprosy Chronic chronic inflammation, systemic sclerosis and hypertrophic scars.

In another embodiment, an avidin fusion protein comprising the amino acid sequence of one of the IL-1R1 antibodies of the invention may be constructed for a variety of purposes. For example, an avidin fusion protein was generated using a mammalian expression vector containing a cDNA sequence encoding a recombinant chicken avidin adjacent to multiple cloning sites for insertion of specific target gene fusion partners. The vector may comprise an avidin sequence having an endogenous signal sequence to enable secretion of an isolated fusion gene partner that does not naturally contain a signal sequence. The fusion protein expressed by the vector has an avidin protein label at the N-terminal portion of the fusion partner. The fusion strategy described herein has the ability to secrete proteins that are generally expressed intracellularly, such as signal transduction genes or nuclear hormone receptors.

Alternatively, the vector can be used to encode avidin without its endogenous signal sequence, which will result in a C-terminal marker of the fusion protein partner. C-terminal avidin fusions also allow for protein secretion based on the endogenous signal sequence of the fusion partner. This strategy provides accurate protein processing and folding or allows the determination of the validity of the desired signal sequence. In addition, the vector may comprise a short nucleotide sequence that encodes an amino acid sequence that can act as a specific enzyme-cutting substrate between the avidin and the fusion partner sequence. This enzyme-cleaving sequence separates the fusion partner from avidin for purification or protein release purposes.

The avidin fusion proteins of the invention can be used, for example, in antibody screening, functional characterization (to determine the usefulness of antibodies as agonists or antagonists, neutralizers, etc.), epitope mapping or immunization strategies. Avidin fusion of the target protein can also be used in the construction of pharmacodynamics, efficacy or other standard assays for testing preclinical or clinical patient samples for the presence of therapeutic antibodies in blood, urine or other tissue samples. Avidin fusion protein partners can be prepared as full length or truncated sequences, as isolated specific structural domains, or as chimeric sequences of fusion partners from other species with other homologs.

Avidin fusion proteins can be expressed using any standard way of introducing genes into cells as described herein and known in the art. Avidin fusion constructs dissolved in lipid solutions, such as lipofectamine (available from Invitrogen, Carlsbad, Calif.), Can be transfected into cells to express proteins, for example, in 293 cells or CHO cells.

Cell lysates from cells expressing conditioned media and / or fusion proteins were collected and supplied to assay substrates such as biotin-coated polystyrene beads or biotin-coated ELISA plates. The conditioned media and / or cell lysate was collected at the point of best expression of the fusion protein. This time point can be determined experimentally by those skilled in the art but is generally about 48 hours after transfection. Fusion proteins can also be analyzed at the cell membrane or can be analyzed intracellularly for expression or binding to a known ligand, receptor or antibody.

The avidin fusion proteins of the invention can be analyzed by known or already characterized methods using biotin-avidin interactions. Such methods include, but are not limited to, flow cytometry and fluorescence imaging / microscopy. For example, avidin fusions expressed in medium or cell lysate can be fed to biotin-coated beads and stained with fluorescently labeled anti-avidin antibodies to express expression levels. Fluorescent antibodies have also been provided to recognize specific fusion protein partners in multicolorimetric assay configurations. Additionally, unlabeled antibodies specific for the fusion protein partner in competition assays can be supplied concurrently with fluorescently labeled antibodies.

In certain embodiments, the present invention provides methods for mapping epitopes using avidin fusion proteins. Examples of epitope mapping methods of the invention are provided below for the mapping of epitopes to anti-IL-1R1 antibodies. However, one skilled in the art will recognize that this method can be readily applied to the mapping of epitopes to all antibodies, which is not limited to IL-1R1 antibodies. For example, a cDNA encoding chicken avidin (having an endogenous signal sequence) fused to a FLAG-labeled sequence at the 3 'end (i.e., a protein recognized by an antibody requiring epitope determination). It can be combined with the 5 'end of. FLAG-labeled fusion genes can be inserted into expression vectors using conventional molecular techniques. Mutant avidin-FLAG marker proteins may be generated using conventional techniques, with certain amino acids substituted (eg, with corresponding amino acid residues from other animal species). Mutant and wild-type proteins can be expressed in host cells and binding of the desired antibody and wild-type or mutant proteins can be detected using, for example, western blotting assays or bead-based binding assays as described herein. . Therefore, epitopes can be defined by measuring the substitution of mutant proteins whose binding with the desired antibody is disrupted.

The following examples, including the experiments performed and the results obtained, are provided for illustrative purposes only and are not intended to limit the invention.

Example 1

Production of Human Monoclonal Antibodies to Interleukin-1 Receptor Type 1 (IL-1R1)

Transgenic HuMab Mouse

Fully human monoclonal antibodies against IL-1 receptor type 1 (IL-1R1) were prepared using the HCo7 strain of transgenic mice expressing human antibody genes. In each of these mouse strains, the endogenous mouse κ light chain gene was homozygous cleaved as described in Chen et al. (1993, EMBO J. 12: 811-820) and the endogenous mouse heavy chain gene was assigned to International Patent Application Publication No. Homozygous cleavage as described in Example 1 of WO 01/09187 (incorporated by reference). Each mouse strain contains KCo5, a human κ light chain transgene, as described in Fishwild et al. (1996, Nature Biotechnology 14: 845-851). HCo7 strains are described in US Pat. No. 5,545,806; No. 5,625,825; It contains the HCo7 human heavy chain transgene, as described in US Pat. No. 5,545,807 (incorporated by reference). HCo7 strain is referred to herein as HuMab mice.

HuMab immunization

To generate fully human monoclonal antibodies against IL-1R1, HuMab mice were purified from purified recombinant IL-1R1 (eg, human IL) derived from an antigenic insect or mammalian cell (eg, CHO cells). The sequence of -1R1 was deposited with GenBank Accession No. NM_000877). General immunization schemes for HuMab mice are described in Lonberg et al. (1994, Nature 368: 856-859; supra from Fishwild et al .; and International Patent Application Publication No. WO 98/24884, each of which theories are cited by reference). Is described in the above). 6-16 week old mice were first injected with antibodies. Purified recombinant preparations (25-50 μg) of IL-1R1 antigen (eg, purified from transfected insect or mammalian cells expressing IL-1R1) were intraperitoneally (IP) or subcutaneous (Sc) HuMab mice were immunized by injection.

HuMab transgenic mice were immunized with complete Freund's adjuvant and antigen dissolved in both injections, followed by IP injection of antigen dissolved in incomplete Freund's adjuvant (up to 11 total immunizations). ). Dozens of mice were immunized with each antigen. A total of 149 mice of HCo7 strain were immunized with IL-1R1. Immune response was confirmed by posterior bleeding.

To select HuMab mice that produce antibodies that bind IL-1R1, the sera of immunized mice were tested by ELISA as described in Fishwild et al., Supra. Briefly, purified recombinant IL-1R1 from insect or mammalian cells was dissolved in PBS at 1-2 μg / mL, coated in microplates at 50 μL / well and incubated overnight at 4 ° C., followed by PBS / Twin (0.05% Blocking with 200 μL / well of 5% chicken serum. Plasma dilutions of IL-1R1-immunized mice were added to each well and incubated for 1-2 hours at ambient temperature. Plates were washed with PBS / Twin and then incubated for 1 hour at room temperature with goat-anti-human IgG Fc-specific polyclonal reagent bound to horseradish peroxidase (HRP). Plates were washed with PBS / Twin and incubated for 1 hour at room temperature with goat-anti-human IgG Fc-specific polyclonal reagent bound to horseradish peroxidase (HRP). After washing, plates were developed with ABTS substrate (Sigma Chemical Company, St. Louis, Missouri, Cat. No. A-1888, 0.22 mg / mL) and spectrophotometrically at an OD of 415-495. Analyzed. Mice with sufficient titers of anti-IL-1R1 human immunoglobulin were used to generate monoclonal antibodies as described below.

Production of Hybridomas Producing Human Monoclonal Antibodies Against IL-1R1

Two days before the sacrifice of mice, antigens were further antigenically stimulated to produce monoclonal antibodies, which were then sacrificed and spleens removed. Mouse splenocytes were isolated from HuMab mice and fused to mouse myeloma cell lines with PEG using standard protocols. In general, 20-30 fusions were generated for each antigen.

Briefly, single cell suspensions of Splenic lymphocytes from immunized mice were treated with P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC Accession No. CRL 1580) or SP2 with 50% PEG (available from Sigma). / 0 non-secreted mouse myeloma cells (ATCC Accession No. CRL 1581) fused with ¼. The cells were plated on a wide bottom microplate at approximately 1 × 10 ⁵ / well and then DMEM supplemented with 10% fetal bovine serum, 10% P388D1 (ATCC Accession No. CRL TIB-63) -conditioned medium, 5 mM HEPES. Obtained from MediaTech, catalog number CRL 10013, 3-5% origen (origen, available from IGEN) dissolved in high glucose, L-glutamine and sodium pyruvate, 0.055 mM 2-mercaptoethanol, 50 mg It was incubated for about 2 weeks in selection medium containing / mL of gentamycin and 1 × HAT (available from Sigma, catalog number CRLP-7185). After 1-2 weeks, the cells were cultured in medium in which the HAT was replaced with HT.

The resulting hybridomas were screened for the production of antigen-specific antibodies. Each well was screened by ELISA (described above) against human anti-IL-1R1 monoclonal IgG antibodies. When growing hybridomas on a large scale, medium was generally identified after 10-14 days. Anti-IL-1R1 monoclonal antibodies were subcloned at least 2 times with limiting dilution if antibody-secreting hybridomas were replated and screened again and still positive for human IgG. Stable subclones were incubated in tissue culture medium for characterization to produce small amounts of antibody.

Screening for Human Monoclonal Antibodies That Bind to IL-1R1

The ELISA assay described above was used to screen hybridomas that showed positive reactivity with the IL-1R1 immunogen. Hybridomas secreting monoclonal antibodies that bind IL-1R1 with high binding activity were subcloned and further characterized. One clone from each hybridoma maintaining parental reactivity was selected to create 5-10 vial cell banks stored in liquid nitrogen.

Isotype-specific ELISAs were performed to determine the isotype of the monoclonal antibodies produced as described herein. In this experiment, 1 μg / mL of mouse-anti-human κ light chain solution dissolved in PBS was coated on a microplate with 50 μL / well and incubated at 4 ° C. overnight. After blocking with 5% chicken serum, the plates were reacted with supernatants from each test monoclonal antibody and purified isotype control. The plate was incubated for 1-2 hours at ambient temperature. Wells were then reacted with human IgGl, IgG2 or IgG4-specific horseradish peroxidase-binding goat anti-human polyclonal antiserum and plates developed and analyzed as follows.

Purified monoclonal antibodies from hybridoma supernatants that bind IL-1R1 as detected by ELISA were obtained in vitro binding assays and human chondrocytes (Cell Applications Inc., San Diego, Calif.). ) And whole blood cell-based assays were used to further test for biological activity. The antibodies showing the best activity were named 15C4, 26F5, 27F2, 24E12 and 10H7. Antibodies were subjected to a preliminary epitope sorting experiment. ELISA plates were coated with human sIL-1R1 (1 + 2 + 3 domains), truncated human sIL-1R1 (1 + 2 domains), rat sIL-1R1, human sIL-1R type 2 and ovalbumin (negative control) It was. Antibody binding was detected with wasabi peroxidase-binding anti-human Fc antibody (obtained from Pierce Chemical Company, Rockford, Ill.). The results are shown in Table 2. "√" in Table 2 indicates a positive result for binding; "X" represents a negative result. Antibodies 15C4, 26F5, 27F2 and 24E12 bind only to the IL-1R1 protein with all three extracellular domains, indicating that each epitope is in the third domain. Antibody 10H7 binds to both the full-length extracellular domain IL-1R1 and truncated proteins having only domains 1 and 2, demonstrating that the epitope for this antibody is present in either domain 1 or 2. There were no test antibodies that cross-react with human type 2 receptors or rat IL-1R1.

Example 2

In Vitro Inhibition of IL-1 Receptor Type 1 Complex Formation by Anti-IL-1R1 Antibody

The ability of antibodies to inhibit the extracellular binding required for IL-1 signaling was assessed with recombinant proteins in an in vitro assay where IL-1 binding to IL-1R forms a high affinity binding site for IL-1RAcP. IL-1 Binding The binding of IL-1RAP to IL-1R (also referred to as “complex formation”) was determined as follows. Recombinant proteins were incubated with binding assays on microplates lacking (control) or present antibodies. IC ₅₀ values were obtained by comparing control values with those obtained from antibodies present at concentrations between 10 fM and 1 μM. Briefly, the assay was performed as follows. Biotinylated IL-1R1 and streptavidin-coated beads (available from Dynal, Dynabiz M-28) were dispensed into microplates. The antibodies were then serially diluted to a wide range of concentrations and added to the appropriate wells. IL-1β or IL-1α was added at a concentration of 1 nM and IL1RAcP labeled with ruthenium (prepared with NHS-labeled (IGEN) according to IGEN protocol) was added to a final concentration of 5 nM. After incubation for 1 hour at room temperature, the binding reaction was analyzed with one of the ORIGEN ™ 1.5 or M8 instruments (available from IGEN International, Inc.). IL-1 Binding IL-1RAcP binding to IL-1R1 was measured by detecting electrochemiluminescent signals associated with IL-1R1 binding beads. The reduction in signal resulting from antibody competition of either IL-1 or IL-1RAcP binding was calculated as a percentage of the ECL signal relative to maximum binding (uncompetitive).

Inhibition response curves for each antibody in this binding assay were established and IC ₅₀ was obtained using PRISM ™ software. Inhibition of IL-1β due to binding is shown in the graph of FIG. IC ₅₀ values for inhibition of complex formation are shown in Table 3 below. Antibodies 15C4, 26F5, 27F2 and 24E12 strongly inhibited complex formation. Such antibodies bind to the third domain of all IL-1R1 as described above. Antibody 10H7 is included in the class of antibodies that bind to the composition of IL-1R lacking a third domain. In the IL-1 induced binding of IL-1RAcP, 10H7 is an inhibitor that is less effective than an antibody binding to the third domain. Inhibition of complex formation by the antibodies of the invention was compared to inhibition by IL-1ra. Antibodies that bind to the third domain had similar or slightly higher ability to inhibit complex formation compared to IL-1ra.

FIG. 13 shows the ability of antibody 15C4 to inhibit IL-1R1 / IL-1α / RAcP complex formation. IC ₅₀ for IL-1R1 / IL-1α / RAcP complex formation is 43 pM.

Example 3

Anti-IL-1R1 Antibodies Inhibit Binding of IL-1β and IL-1ra to Receptors

The ability of anti-IL-1R1 antibodies to inhibit the binding of either IL-1β or IL-1ra to IL-1R1 was assessed in an assay with recombinant protein. The reaction mixture comprises 0.1 mg / mL Dynavid M-280 streptavidin (available from Dinal) and 1 nM of biotinylated IL-1R1. To this the antibody was added at a concentration of 320 nM to 0.3 nM. Ruthenium-labeled IL-1β (5 nM) or IL-1ra (1 nM) was added and left for 1 hour at room temperature to allow binding to occur. The reaction mixture was measured as above using the ORIGEN ™ 1.5 or M8 instrument (available from IGEN International, Inc.). The percentage of competition was expressed as a percentage of the ECL signal for maximum binding (uncompetitive). The most effective antibodies, antibodies 15C4, 26F5 and 27F2, blocked ligand binding to receptor (IL-1β) but did not significantly inhibit binding of IL-1ra compared to IgG controls. In contrast, antibody 24E12 bound to the receptor but did not block binding of IL-1β or IL-1ra to the receptor. Therefore, antibody 24E12 represents the only class of third domain binding antibodies that is different from the class represented by 15C4, 26F5 and 27F2. Antibody 10H7 inhibits both IL-1β and IL-1ra which bind to the receptor. The results are shown in FIG.

Example 4

Chondrocytes and Human Whole Blood Assay

Primary human chondrocytes (obtained from Cell Applications Inc., San Diego, Calif.) Were subjected to 10,000 cells / well in DMEM medium containing 1% PBS and 1% Pen Strep (Gibco). Inoculated in 96-well plates by density. Cells were regenerated overnight before adding the anti-IL-1R1 antibody for 20 minutes at concentrations ranging from 10 nM to 0.1 pM. IL-1β was added at a concentration of 1 pM (˜EC ₅₀ ) and incubated at 37 ° C. for 16 hours to obtain a culture supernatant. IL-6 levels in the supernatants were measured using an ELISA (obtained from Pierce-Endogen, Rockford, Ill., Catalog number EH2IL-65) according to the manufacturer's instructions. Inhibition response curves for each antibody of the invention in cell-based assays were established and IC ₅₀ values were obtained using PRISM ™ software. Antibodies 15C4, 26F5 and 27F2 are more effective inhibitors of IL-1 signaling compared to IL-1ra (FIG. 15A). Antibodies 24E12 and 10H7 show significantly less effect than 15C4 and 27F2 (FIG. 15B). IC ₅₀ values for inhibition of IL-1β induced IL-6 producing human chondrocytes are shown in Tables 4A and 4B (corresponding to FIGS. 15A and 15B, respectively).

Anti-IL-1R1 monoclonal antibodies 15C4, 26F5 and 27F2 were pre-incubated for 40-60 minutes with human whole blood obtained from normal volunteers by a heparin sodium vacuum vessel. The assay proceeded as follows: 100 μL of fresh blood separated was equally divided into wells of a 96-well plate. 50 μL of antibody was added by dissolving in RPMI medium containing 100% human AB serum. IL-1β was then added at a concentration of 30 pM (EC ₅₀ ). After 18 hours culture supernatants were obtained and IL-6 levels in the supernatants were measured using ELISA. As a control IL-1ra was pre-incubated for 40-60 minutes with whole blood and IL-6 production was measured as above. Three anti-IL-1R1 antibodies inhibited IL-1 activity more effectively than IL-1ra (FIG. 16). IC ₅₀ values for inhibition of IL-1-induced IL-6 production in human whole blood are shown in Table 5.

Example 5

Mutagenesis and Epitope Mapping

The mutant proteins ( "muteins") substituted with a portion of the IL-1R1-specific mutagenesis (obtained from Promega located at a Altered Sites ^® In vitro mutagenesis system, Wisconsin) as a human sequence which is rat amino acid residues corresponding manufacturing It was. Fifteen different mutant plasmids were constructed (see the bold lines numbered in FIG. 17). Plasmids encoding these substituted proteins and the original IL-1R1 were transiently transfected into CHO cells (ATCC Accession No. CRL-9096). Transfectants with nothing transfected were prepared as negative controls. Conditioned medium (CM) from these cells was concentrated up to 20-fold using a Centrirep 10 concentration column (available from Amicon). Mutein expression was measured by SDS-PAGE and Western blotting. Thirteen mutant proteins were expressed at the level at which antibody binding occurred. Proteins were loaded into gels, electrophoresed and transferred to membranes. Membranes were blocked with 1% milk dissolved in PBS, 0.1% Tween-20, followed by 0.5 ug / mL anti-IL-1R antibody 15C4, 27F2 or 24E12 dissolved in PBS and 0.1% Tween-20 for 1 hour at room temperature. It was incubated for a while. After washing, the membrane was incubated with goat anti-human IgG-Fc-HRP. The signal was detected using a chemiluminescent (ECL) substrate (obtained from Pierce Chemical Company, Rockford, Ill.). Human specific sequences critical for antibody binding were identified as human sequences substituted with rat sequences with reduced or eliminated ECL signals. 15C4 recognition of mutations 1, 2, 4 and 10 was lower compared to 24E12 (FIG. 18, first part). Likewise, binding of 27F2 to mutations 1, 2 and 4 was also low (FIG. 18, middle). 24E12 had significantly lower binding to mutations 12, 13, 14 and 15 (FIG. 18, last part).

Isolation and characterization of human anti-IL-1R1 identified three different classes of competitive antibodies (FIG. 19). The strongest inhibitor of IL-1 biological activity, validated by cell-based bioassay, is the antibody that binds to the third domain of IL-1R1 and prevents IL-1-β binding. Epitope mapping experiments using a third domain mutant protein demonstrated that antibodies of this class, including 15C4, 27F2 and 26F5, share overlapping epitopes but do not share the same morphological epitopes. 20 and 21 show the location of the 15C4 epitope on the third domain of IL-1 receptor in a ribbon diagram of IL-1ra binding IL-1 receptor (Schreuder et al., 1997, Nature 386: 194-200). will be. IL-1 receptor residues that define the binding of the most effective class of antibodies are shown in gray. Since such antibodies show excellent potency, this epitope is defined as the binding site for antibodies of the class with excellent potency. The 15C4 and 27F2 binding sites overlapped but were not identical, as determined by mutation analysis of 15 different sites in IL-1R1 described above. This site is depicted as thick lines numbered above the protein sequence in FIG. 17. The critical regions of the interaction were mutations at site 1 (LSDIA; SEQ ID NO: 41), site 2 (VIDE; SEQ ID NO: 42), site 4 (YSV) and site 10 (TCFA; SEQ ID NO: 43). Appear within. 15C4 and 27F2 binding sites are included within site 1 and site 2 because binding to human sites is replaced by rat residues in either of these sites. 27F2 differs from 15C4 in that the change at site 4 completely blocks the binding while the binding of 15C4 is reduced but not completely blocked. Mutant 10 also reduced 15C4 binding but 27F2 did not show an obvious interaction with this site. Experiments with the crystal structure show that it defines the surface of the third domain that these residues are oriented towards the space occupied by the bound ligand (FIGS. 20 and 21) (Vigers et al., 1997, Nature 386: 190-194).

The second class of antibodies identified, 10H7, does not require a third domain for binding and, unlike the preferred class, inhibits IL-1ra binding. This class is active in bioassays but exhibits less efficacy than the preferred class.

In contrast to the strong inhibition of IL-1 bioassays with the preferred class of antibodies, 24E12 is not an effective inhibitor in bioassays. Antibody 24E12 inhibits IL-1RAcP binding with IL-1-binding IL-1R. The epitopes of this class of antibodies defined by mutations 12, 13, 14 and 15 are proximal to the domain of the transmembrane of IL-1R1 and are present in a region directly related to either IL-1 or IL-1ra binding ( 22).

Example 6

Cloning of Anti-IL-1R1 Antibody Heavy and Light Chains

Cloning of Anti-IL-1R1 15C4 MAb Light Chain

Light chains for three hybridomas expressing the αIL-1R1 binding monoclonal antibodies 15C4, 27F2 and 26F5 were obtained from the mammalian cell expression vector pDSRα19 (available from Amgen Incorporated, an international patent incorporated herein by reference). See Publication No. WO 90/14363). The construction of the plasmid encoding the 15C4 κ light chain is clearly described herein; Cloning of other light chain species was performed using a similar method. The αIL-1R1 light chain variable region was prepared using a polymerase chain reaction (PCR) amplification method from first strand DNA prepared from αIL-1R1 hybridoma 15C4 total RNA prepared using TRIzol ^® reagent (available from Invitrogen). Obtained. The first strand cDNA was synthesized using random primers with an extension adapter (5'-GGC CGG ATA GGC CTC CAN NNN NNT-3 '; SEQ ID NO: 44) and 5' RACE (acute at the cDNA terminus). Amplification) was performed using the GeneRacer ™ kit (available from Invitrogen). In the complete light chain, the forward primer is GeneRacer ™ containing primer (5'-GGA CAC TGA CAT GGA CTG AAG GAG TA-3 '; SEQ ID NO: 45) and the reverse primer is 5'-GGG GTC AGG CTG GAA CTG AGG-3 '(SEQ ID NO: 46). RACE products were cloned into pCR4-TOPO (available from Invitrogen) and DNA sequences were determined. The 15C4 κ chain consensus DNA sequence was used to design primers for full length antibody chain PCR amplification. The 5 ′ κ PCR primer encodes the amino terminus of the signal sequence, the XbaI restriction enzyme site and the optimized Kozak sequence (5 ′ CAG CAG AAG CTT CTA GAC CAC CAT GTC GCC ATC ACA ACT CAT TGG G-3 ′; SEQ ID NO : 47). The 3 'primers encode the SalI restriction site as well as the carboxy terminal and termination codons (5'-CTT GTC GAC TCA ACA CTC TCC CCT GTT GAA GCT C-3'; SEQ ID NO: 48).

Full length αIL-1R1 15C4 κ chain clones were obtained using pCR4: 15C4 κ clones by PCR amplification with 5 ′ and 3 ′ αIL-1R1 15C4 κ primers. A 733 bp product encoding 233 amino acid residues (including 19 amino acid κ chain signal sequences) of the αIL-1R1 15C4 κ chain was prepared by PCR. PCR products were purified using QIAquick PCR Purification Kit (Qiagen Catalog No. 28104), digested with XbaI and SalI, gel separated and purified using QIAquick Gel Extraction Kit (Qiaquin Catalog No. 28704). This PCR fragment containing the complete αIL-1R1 15CA κ chain was then bound into the mammalian expression vector pDSRα19. 15C4 κ chain expression clones were DNA sequenced to confirm that they encode the same peptide identified in the 15C4 hybridoma. The final expression vector, pDSRα19: 15C4 κ, is 5468 bp and contains seven functional regions listed in Table 6.

pDSR: hIgG1C _H  The composition of

pDSRα19: Rat variable region / human constant region IgG1 (rVh / hCh1) MAb expression plasmids were subjected to XbaI and BsmBI terminal rat antibody variable region PCR products, human IgG1 constant regions induced by SalI cleavage (C _H1 , hinge, C _H2 and C) _H3 domain) and linear plasmid pDSRα19: constituted by combining three pieces of gel isolates of BsmBI and SalI fragments from linearized pDSRα19 with hIgG1 C _H (HindIII and BsmBI termini) and XbaI and SalI termini (co-owned and co-owned) -See US Provisional Application No. 60 / 370,407, "Human Anti-OPGL Neutralizing Antibodies as Selective OPGL Pathway Inhibitors," filed April 5, 2002). The final expression vector, pDSRα19: rat variable region / human constant region IgG1 (rVh / hCh1), is 6158 bp and contains seven functional regions described in Tables 7A and 7B.

To remove the rat variable region, the pDSR19: rat variable region / human constant region IgG1 plasmid was cut with restriction enzymes XbaI and BsmBI to prepare a linear plasmid pDSRα19: hIgG1C _H which was purified using a QIAquick gel extraction kit. The linear plasmid pDSRα19: hIgG1C _H containing 1 kbp human IgG1 constant region domains was used to obtain hybridoma derived αIL-1R antibody variable regions.

Cloning of Anti-IL-1R1 15C4 MAb Heavy Chain

The heavy chains for 10 hybridomas expressing αIL-1R1 binding monoclonal antibodies 15C4, 27F2 and 26F5 were cloned into mammalian cell expression vector pDSRα19. The construction of the plasmid encoding the 15C4 heavy chain was clearly described; Cloning of other heavy chain species was performed using a similar method. The TRIzol ^? IL-1R1 15C4 heavy chain variable region ^? Prepared from the first strand cDNA prepared from αIL-1R1 hybridoma 15C4 total RNA prepared using reagents using the PCR amplification method. The first strand cDNA was synthesized using random primers with extension linkers (5'-GGC CGG ATA GGC CTC CAN NNN NNT-3 '; SEQ ID NO: 44) and 5' RACE (acute amplification of cDNA ends) was generated by GeneRacer. It was performed using the ™ kit. In the heavy chain of partial length, the forward primer is GeneRacer ™ inclusion primer (5'-GGA CAC TGA CAT GGA CTG AAG GAG TA-3 '; SEQ ID NO: 45) and the reverse primer is 5'-TGA GGA CGC TGA CCA CAC G -3 '(SEQ ID NO: 52). RACE products were cloned into pCR4-TOPO and DNA sequences were determined. The 15C4 heavy chain variable region consensus DNA sequence was used to design primers for heavy chain variable region PCR amplification. The 5 'heavy chain PCR primer encodes the amino terminus of the signal sequence, the XbaI restriction enzyme site and the optimized Kozak sequence (5'-CAG CAG AAG CTT CTA GAC CAC CAT GGG GTC AAC CGC CAT CCT CG-3'; SEQ ID NO : 53). The 3 'primer encodes the carboxy terminus of the variable region comprising the naturally occurring sense strand BsmBI site (5'-GTG GAG GCA CTA GAG ACG GTG ACC AGG GTT CC-3'; SEQ ID NO: 54).

Construction of Anti-IL-1R1 IgG1 Heavy Chain Expression Clone

Full length αIL-1R1 15C4 heavy chain clones were obtained from pCR4: 15C4 heavy chain clones by PCR amplification with 5 ′ and 3 ′ αIL-1R1 15C4 heavy chain primers. A 442 bp product encoding 137 amino acid residues (including 19 amino acid heavy chain signal sequences) of the αIL-1R1 15C4 heavy chain variable region was generated by PCR reaction. PCR products were purified using QIAquick PCR Purification Kit, digested with XbaI and BsmBI, gel separated and purified using QIAquick Gel Extraction Kit. This fragment containing the complete αIL-1R1 15CA heavy chain variable region was then bound into the mammalian expression vector pDSRα19: hIgG1C _H. 15C4 heavy chain IgG1 expressing clones were DNA sequenced to confirm that they encode the same heavy chain variable region peptides identified in 15C4 hybridomas. The final expression vector, pDSRα19: 15C4 IgG1 heavy chain, is 6173 bp and contains the seven functional regions described in Table 8.

pDSRα19: hIgG2C _H  The composition of

pDSRα19: Human variable region / human constant region IgG2 (hVh / hCh2) MAb expression plasmids were subjected to XbaI and BsmBI terminal human antibody variable region PCR products, human IgG2 constant region with BsmBI and SalI termini (C _H1 , hinge, C _H2 and C) _H3 domain) PCR product and 3-fragment of linearized pDSRα19 with XbaI and SalI ends. PDSRα19, the final expression vector: human variable region / human constant region IgG1 (hVh / hCh2) (US Provisional Application No. 60 / 370,407, filed April 5, 2002, co-owned and co-pending, “Optional OPGL Human anti-OPGL neutralizing antibody as a pathway inhibitor ”is 6164 bp and contains seven functional regions described in Tables 9A and 9B.

To remove the human variable region, the pDSRα19: human variable region / human constant region IgG2 plasmid was digested with restriction enzymes XbaI and BsmBI to prepare a linear plasmid pDSRα19: hIgG2C _H and purified using a QIAquick gel extraction kit. A linear plasmid pDSRα19: hIgG2C _H containing 1 kbp of human IgG2 constant region domains was used to obtain a hybridoma derived αIL-1R antibody variable region.

Construction of Anti-IL-1R1 IgG2 Heavy Chain Expression Clone

The αIL-1R1 15C4 heavy chain variable region fragments described above were bound into mammalian expression vector pDSRα19: hIgG2C _H. 15C4 heavy chain IgG2 expressing clones were DNA sequencing to confirm that they encode the same heavy chain variable region peptides identified within 15C4 hybridomas. The final expression vector, pDSRα19: 15C4 IgG2 heavy chain, is 6161 bp and contains the seven functional regions listed in Table 10.

pDSRα19: hIgG4C _H  The composition of

3-fragment of linearized pDSRα19 with XbaI and BsmBI terminal human antibody variable region PCR products, gel isolated BsmBI and SalI cleaved human IgG4 constant region (C _H1 , hinge, C _H2 and C _H3 domains) fragments and XbaI and SalI termini PDSRα19: human variable region / human constant region IgG4 (hVh / hCh4) MAb expression plasmids were combined. PDSRα19, the final expression vector: human variable region / human constant region IgG4 (hVh / hCh4) (US Provisional Application No. 60 / 370,407, filed April 5, 2002, co-owned and co-pending, “Optional OPGL Pathway” Human anti-OPGL neutralizing antibody as inhibitor ”is 6167 bp and contains the seven functional regions described in Tables 11A and 11B.

To remove the human variable region, the pDSR19: human variable region / human constant region IgG4 plasmid was digested with restriction enzymes XbaI and BsmBI to prepare a linear plasmid pDSRα19: hIgG4C _H which was purified using a QIAquick gel extraction kit. A linear plasmid pDSRα19: hIgG4C _H containing 1 kbp human IgG4 constant region domains was used to obtain hybridoma derived αIL-1R antibody variable regions.

Construction of Anti-IL-1R1 IgG4 Heavy Chain Expression Clone

The αIL-1R1 15C4 heavy chain variable region fragment described above was bound into mammalian expression vector pDSRα19: hIgG4C _H. 15C4 heavy chain IgG4 expressing clones were DNA sequenced to confirm that they encode the same heavy chain variable region peptides identified in the 15C4 hybridomas. The final expression vector, pDSRα19: 15C4 IgG4 heavy chain, is 6164 bp and contains the seven functional regions described in Table 12.

Example 7

Expression of Anti-IL-1R1 Antibody in Chinese Hamster Ovary (CHO) Cells

Recombinant anti-IL-1R1 antibodies are generated in Chinese hamster ovary cells, particularly CHO AM-1 / D (available from Amgen Inc.), as described in US Pat. No. 6,210,924, which is incorporated herein by reference. It was. Briefly, DNA sequences encoding the complete heavy or light chain of each anti-IL-1R1 antibody of the invention were cloned into expression vectors. An expression vector capable of expressing a full heavy chain and an expression vector expressing the complete light chain of the appropriate anti-IL-1R1 antibody were co-transfected into CHO AM-1 / D cells. For example, to generate a 26F5 antibody, a vector capable of expressing a complete light chain comprising the amino acid sequence as described in SEQ ID NO: 38 and SEQ ID NO: 20, SEQ ID NO: 22 or SEQ ID NO: Vectors capable of expressing a complete heavy chain comprising the amino acid sequence described in 24 were co-transfected into the cells. To generate a 27F2 antibody, a vector capable of expressing a complete light chain comprising an amino acid sequence as described in SEQ ID NO: 38 and an amino acid as described in SEQ ID NO: 26, SEQ ID NO: 28 or SEQ ID NO: 30 Vectors capable of expressing a complete heavy chain comprising the sequence were co-transfected into the cells. To generate a 15C4 antibody, a vector capable of expressing a complete light chain comprising an amino acid sequence as described in SEQ ID NO: 40 and an amino acid as described in SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36 Vectors capable of expressing a complete heavy chain comprising the sequence were co-transfected into the cells. Table 13 shows the complete heavy and complete light chains of the various IL-1R1 antibodies. The notation "... / IgG_" describes the sequence of the constant region of a particular antibody.

Stable expression of anti-IL-1R1 antibodies was achieved by co-transfecting expression vectors into dehydrofolate reductase deficient (DHFR ⁻ ) CHO AM-1 / D cells. Transfection was performed using standard techniques (calcium phosphate co-precipitation) and DHFR screening. Transfected colonies were isolated and grown in aggregate in 24-well plates. Antibodies produced by the transfected cells were tested for proper folding and neutralizing activity. Clones that overproduce properly folded anti-IL-1R1 antibodies of the IgG1, IgG2 and IgG4 isotypes were selected and the antibodies were purified as follows.

Example 8

Production of Anti-IL-1R1 Antibodies

Anti-IL-1R1 antibody was produced by expression in clones of CHO cells. In each production, cells from one vial were lysed in serum-free cell culture medium. Cells were initially grown continuously in successive Spinner flasks until they were incubated in T-flasks and produced an inoculum suitable for inoculation into a 20 L bioreactor. After growing for 5-10 days, the culture was used to inoculate a 300 L bioreactor. After further growing for 5-10 days, the culture was used to inoculate a 2000 L bioreactor. Production was carried out in a 2000 L bioreactor using fed-batch culture and nutrient feed containing concentrated media components was added to maintain cell growth and culture viability. Production continued for about 2 weeks, which is the time when anti-IL-1R1 antibody was produced structurally by the cells and secreted into the cell culture medium.

The production reactor was adjusted to pH, temperature and dissolved oxygen levels: pH was adjusted by addition of carbon dioxide gas and sodium carbonate; Dissolved oxygen was controlled by air, nitrogen and oxygen gas flows.

At the end of production, the cell culture was fed into a disk stack centrifuge and the culture supernatant was separated from the cells. The concentrate was passed through a depth filter and then through a 0.2 μm filter to make it more transparent. The cleared conditioned media was then concentrated by tangential flow ultrafiltration. The conditioned medium was concentrated 15-30 times. The resulting concentrated conditioned medium was purified or frozen for later purification.

Example 9

Epitope Mapping with Avidin-Fusion Proteins

To produce an avidin-fusion protein, a cDNA encoding chicken avidin (with an endogenous signal sequence) encoding a mature exogenous domain of human or cynomolgus IL-1R1 fused to the FLAG-labeled sequence at the 3 'end. Binding to the 5 'end of cDNA. FLAG-labeled fusion genes were bound into pALTERMAX vectors (available from Promega Corporation) using conventional molecular techniques. The amino acid sequence of the avidin-human IL-1R1 fusion protein is shown in FIG. 23 (SEQ ID NO: 59). The amino acid sequence of the avidin-cynomolgus IL-1R1 fusion protein is shown in FIG. 24 (SEQ ID NO: 60). The corresponding no-IL-1R1-FLAG proteins between avidin mutant of the human amino acid substitution in between nomol Goose residues was generated using the Altered Site ^® Ⅱ mammal in vitro Mutagenesis System (available from Promega Corp.). Mutations are shown in FIG. 24.

Plasmids encoding avidin-cynoIL-1R mutations and wild-type proteins, as well as avidin huIL-1R1 FLAG protein, were prepared using cytofectin transfection reagent (available from Bio-Rad Laboratories, Inc.). (Typically available standard human kidney cell line). A transfectant with nothing transfected was used as a negative control. Anti-huIL-1R1 monoclonal antibody (MAb) that binds to these proteins was evaluated by Western blotting and bead-based binding assays using conditioned media (CM) harvested from transfected cells.

In Western blotting assay, CM was diluted 1: 3 in non-reducing SDS sample buffer, boiled for 5-10 minutes and loaded onto 10% tris-glycine gel. After SDS-PAGE and Western metastasis the membrane was blocked with 3% BSA / 1% ovalbumin dissolved in PBS / 0.1% Tween-20 (PBST) and stained with anti-huIL-1R1 MAb. Goat anti-human IgG-Fc-HRP antibody (obtained from Pierce Chemical Company) diluted 1: 1 to PBST was used for secondary detection. Anti-FLAG detection was used to standardize protein loading. Image capture and densitometry were performed using a FluorChem 8000 digital imaging system (available from Alpha Innotek Corporation). Signal intensity for anti-huIL-1R1 MAb was normalized to the values for anti-FLAG antibodies to account for variations in protein loading. Antibody binding was expressed as a percentage of binding to avidin-human IL-1R1-FLAG.

The results of western blotting are shown in Figure 25A. 25B shows densitometry analysis of two western blotting tests. Human residues critical for antibody binding are those residues whose signal is restored when substituted into cynoIL-1R1. In general, binding to many antibodies (15C4 / IgG2, 5B8, 1C2, 24H2, 16E9, 24E4, and 20G1), alone or in combination, in mutations 1 and 2 (shown in FIG. 24), restored mutations 10.1 and 10.2. Did. None of these antibodies bound wild type cynoIL-1R1. Both antibodies (27F2 and 19C8) consistently bound not only wild type cynoIL-1R1 but all mutant proteins. This suggests that epitope 4 (residues Y279-V281 of cynoIL-1R1), which is identified in rat / paralog protein and has not changed in cynomolgus IL-1R1, is the dominant epitope for this antibody. Epitope 4 is shown in bold italics underlined in the amino acid sequence shown in FIG. 24.

In a multiplexed bead-based binding assay, avidin fusion proteins were obtained by incubation with biotin-coated fluorescent beads in CM and were one bead per fusion protein (Bidlite Multi-Biotin 10Flex Bead Kit; obtained from Upstate Biotechnology ). Beads were washed, pooled in PBST and dispensed equally into each well of a 96-well filter bottom plate (available from Millipore Corporation). Antibody (anti-huIL-1R1 MAb or anti-FLAG MAb) was added at 25 μg / ml and incubated for 1 hour. The beads were washed again and a mixture of phycoerythrin-binding anti-mouse IgG antibody and anti-human IgG (Fab ') 2 was used to detect antibody binding. After incubation for 1 hour, the beads were washed and resuspended in PBST. Average phosphor intensity (MFI) was measured using Luminex 100 (available from Luminex Corporation). Data was normalized using MFI values for anti-FLAG MAb binding to account for variations in protein loading. Antibody binding was expressed as a percentage of binding with avidin-huIL-1R1-FLAG (FIG. 26). The binding pattern of anti-IL-1R1 antibody against wild type cynomolgus and human IL-1R1 protein as well as avidin-cynoIL-1R1-FLAG protein mutated with human residues is consistent with the immunoblot analysis shown in FIG. 25. do.

It is to be understood that the above specification highlights certain embodiments of the invention and that all variations or alternative equivalents thereof are included within the spirit and scope of the invention as set forth in the appended claims.

SEQUENCE LISTING <110> Varnum, Brian       Witte, Alison       Vezina, Chris       Wong, Lu Min       Qian, Xueming <120> Therapeutic Human Anti-IL-1R Monoclonal Antibody <130> 01,1554 <160> 79 <170> PatentIn version 3.0 <210> 1 <211> 990 <212> DNA <213> Homo Sapiens <400> 1 gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctat agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 2 <211> 330 <212> PRT <213> Homo sapiens <400> 2 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys             100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro         115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys     130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu                 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu             180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn         195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly     210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr                 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn             260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe         275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn     290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 330 <210> 3 <211> 321 <212> DNA <213> Homo Sapiens <400> 3 cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t 321 <210> 4 <211> 107 <212> PRT <213> Homo sapiens <400> 4 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe             20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln         35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser     50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser                 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys             100 105 <210> 5 <211> 978 <212> DNA <213> Homo sapiens <400> 5 gcctccacca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag 60 agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc 240 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 300 aaatgttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 360 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgc 420 gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 480 gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttccgt 540 gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga gtacaagtgc 600 aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 660 cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 720 caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 780 gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac 840 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 900 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 960 tccctgtctc cgggtaaa 978 <210> 6 <211> 326 <212> PRT <213> Homo sapiens <400> 6 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro             100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp         115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp     130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn                 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp             180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro         195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu     210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile                 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr             260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys         275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys     290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys                 325 <210> 7 <211> 981 <212> DNA <213> Homo Sapiens <400> 7 gccagcacca aggggccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag 60 agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 240 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 300 aaatatggtc ccccatgccc atcatgccca gcacctgagt tcctgggggg accatcagtc 360 ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 420 tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 480 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 540 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 600 tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 660 gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 720 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 780 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 840 gacggctcct tcttcctcta cagcaggcta accgtgraca agagcaggtg gcaggagggg 900 aatgtcttct catgctccgt gakgcatgag gctctgcaca accactacac acagaagagc 960 ctctccctgt ctctgggtaa a 981 <210> 8 <211> 327 <212> PRT <213> Homo sapiens <400> 8 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr             20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser         35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser     50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys                 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro             100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys         115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val     130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe                 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp             180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu         195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg     210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp                 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys             260 265 270 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser         275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser     290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys                 325 <210> 9 <211> 417 <212> DNA <213> Homo Sapiens <400> 9 atggagtttg ggctgagctg ggtcttcctc gttgctcttt taagaggtgt ccagtgtcag 60 gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120 tgtgcagcgt ctggattcac cttcagcaac tatggcatgc actgggtccg ccaggctcca 180 ggcaaggggc tggagtgggt ggcaggcatt tggaatgatg gaattaataa ataccatgca 240 cactccgtga ggggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300 caaatgaaca gcccgagagc cgaggacacg gctgtgtatt actgtgcgag agcacggtct 360 ttcgactggc tattatttga gttctggggc cagggaaccc tggtcaccgt ctctagt 417 <210> 10 <211> 139 <212> PRT <213> Homo sapiens <400> 10 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln             20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe         35 40 45 Ser Asn Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Ala Gly Ile Trp Asn Asp Gly Ile Asn Lys Tyr His Ala 65 70 75 80 His Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Pro Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Ala Arg Ser Phe Asp Trp Leu Leu Phe Glu Phe         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser     130 135 <210> 11 <211> 384 <212> DNA <213> Homo Sapiens <400> 11 atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60 gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 120 ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 180 ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 240 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 300 gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctccgct cactttcggc 360 ggagggacca aggtggagat caaa 384 <210> 12 <211> 128 <212> PRT <213> Homo sapiens <400> 12 Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser             20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser         35 40 45 Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro     50 55 60 Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser                 85 90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser             100 105 110 Asn Trp Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys         115 120 125 <210> 13 <211> 417 <212> DNA <213> Homo Sapiens <400> 13 atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60 gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120 tgtgcagtgt ctggattcac cttcagtaac tatggcatgc actgggtccg ccaggctcca 180 ggcaaggggc tggagtgggt ggcagctata tggaatgatg gagaaaataa acaccatgca 240 ggctccgtga ggggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300 caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag aggacgatat 360 tttgactggt tattatttga gtattggggc cagggaaccc tggtcaccgt ctctagt 417 <210> 14 <211> 139 <212> PRT <213> Homo sapiens <400> 14 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln             20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe         35 40 45 Ser Asn Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Ala Ala Ile Trp Asn Asp Gly Glu Asn Lys His His Ala 65 70 75 80 Gly Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Trp Leu Leu Phe Glu Tyr         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser     130 135 <210> 15 <211> 411 <212> DNA <213> Homo Sapiens <400> 15 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgag 60 gtgcagctga tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc 120 tgtaagggtt ctggatacag cttttccttc cactggatcg cctgggtgcg ccagatgccc 180 gggaaaggcc tggagtggat ggggatcatc catcctggtg cctctgatac cagatacagc 240 ccgtccttcc aaggccaggt caccatctca gccgacaact ccaacagcgc cacctacctg 300 cagtggagca gcctgaaggc ctcggacacc gccatgtatt tctgtgcgag acaaagggaa 360 ctcgactact ttgactactg gggccaggga accctggtca ccgtctctag t 411 <210> 16 <211> 137 <212> PRT <213> Homo sapiens <400> 16 Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu Ala Val Leu Gln Gly 1 5 10 15 Val Cys Ala Glu Val Gln Leu Met Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe         35 40 45 Ser Phe His Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu     50 55 60 Glu Trp Met Gly Ile Ile His Pro Gly Ala Ser Asp Thr Arg Tyr Ser 65 70 75 80 Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Asn Ser Asn Ser                 85 90 95 Ala Thr Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met             100 105 110 Tyr Phe Cys Ala Arg Gln Arg Glu Leu Asp Tyr Phe Asp Tyr Trp Gly         115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser     130 135 <210> 17 <211> 378 <212> DNA <213> Homo Sapiens <400> 17 atgtcgccat cacaactcat tgggtttctg ctgctctggg ttccagcctc caggggtgaa 60 attgtgctga ctcagtctcc agactttcag tctgtgactc caaaggagaa agtcaccatc 120 acctgccggg ccagtcagag cattggtagt agcttacact ggtaccagca gaaaccagat 180 cagtctccaa agctcctcat caagtatgct tcccagtcct tctcaggggt cccctcgagg 240 ttcagtggca gtggatctgg gacagatttc accctcacca tcaatagcct ggaagctgaa 300 gatgctgcag cgtattactg tcatcagagt agtagtttac ctctcacttt cggcggaggg 360 accaaggtgg agatcaaa 378 <210> 18 <211> 126 <212> PRT <213> Homo sapiens <400> 18 Met Ser Pro Ser Gln Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala 1 5 10 15 Ser Arg Gly Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val             20 25 30 Thr Pro Lys Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile         35 40 45 Gly Ser Ser Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys     50 55 60 Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser                 85 90 95 Leu Glu Ala Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser             100 105 110 Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys         115 120 125 <210> 19 <211> 1407 <212> DNA <213> Homo Sapiens <400> 19 atggagtttg ggctgagctg ggtcttcctc gttgctcttt taagaggtgt ccagtgtcag 60 gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120 tgtgcagcgt ctggattcac cttcagcaac tatggcatgc actgggtccg ccaggctcca 180 ggcaaggggc tggagtgggt ggcaggcatt tggaatgatg gaattaataa ataccatgca 240 cactccgtga ggggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300 caaatgaaca gcccgagagc cgaggacacg gctgtgtatt actgtgcgag agcacggtct 360 ttcgactggc tattatttga gttctggggc cagggaaccc tggtcaccgt ctctagtgcc 420 tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 720 tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 780 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840 gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 900 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 960 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1020 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1080 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 1140 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260 gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1320 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380 aagagcctct ccctgtctcc gggtaaa 1407 <210> 20 <211> 469 <212> PRT <213> Homo sapiens <400> 20 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln             20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe         35 40 45 Ser Asn Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Ala Gly Ile Trp Asn Asp Gly Ile Asn Lys Tyr His Ala 65 70 75 80 His Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Pro Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Ala Arg Ser Phe Asp Trp Leu Leu Phe Glu Phe         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val     210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu                 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr             260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val         275 280 285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val     290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu                 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala             340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro         355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln     370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr                 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu             420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser         435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser     450 455 460 Leu Ser Pro Gly Lys 465 <210> 21 <211> 1395 <212> DNA <213> Homo sapiens <400> 21 atggagtttg ggctgagctg ggtcttcctc gttgctcttt taagaggtgt ccagtgtcag 60 gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120 tgtgcagcgt ctggattcac cttcagcaac tatggcatgc actgggtccg ccaggctcca 180 ggcaaggggc tggagtgggt ggcaggcatt tggaatgatg gaattaataa ataccatgca 240 cactccgtga ggggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300 caaatgaaca gcccgagagc cgaggacacg gctgtgtatt actgtgcgag agcacggtct 360 ttcgactggc tattatttga gttctggggc cagggaaccc tggtcaccgt ctctagtgcc 420 tccaccaagg gcccatcggt cttccccctg gcgccctgct ccaggagcac ctccgagagc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ctctgaccag cggcgtgcac accttcccag ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca acttcggcac ccagacctac 660 acctgcaacg tagatcacaa gcccagcaac accaaggtgg acaagacagt tgagcgcaaa 720 tgttgtgtcg agtgcccacc gtgcccagca ccacctgtgg caggaccgtc agtcttcctc 780 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacgtgcgtg 840 gtggtggacg tgagccacga agaccccgag gtccagttca actggtacgt ggacggcgtg 900 gaggtgcata atgccaagac aaagccacgg gaggagcagt tcaacagcac gttccgtgtg 960 gtcagcgtcc tcaccgttgt gcaccaggac tggctgaacg gcaaggagta caagtgcaag 1020 gtctccaaca aaggcctccc agcccccatc gagaaaacca tctccaaaac caaagggcag 1080 ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag 1140 gtcagcctga cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtgggag 1200 agcaatgggc agccggagaa caactacaag accacacctc ccatgctgga ctccgacggc 1260 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1320 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1380 ctgtctccgg gtaaa 1395 <210> 22 <211> 465 <212> PRT <213> Homo sapiens <400> 22 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln             20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe         35 40 45 Ser Asn Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Ala Gly Ile Trp Asn Asp Gly Ile Asn Lys Tyr His Ala 65 70 75 80 His Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Pro Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Ala Arg Ser Phe Asp Trp Leu Leu Phe Glu Phe         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 155 160 Thr Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val     210 215 220 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys 225 230 235 240 Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro                 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser             260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ser Glu Asp         275 280 285 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn     290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu                 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys             340 345 350 Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr         355 360 365 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr     370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu                 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys             420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu         435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     450 455 460 Lys 465 <210> 23 <211> 1398 <212> DNA <213> homo sapiens <400> 23 atggagtttg ggctgagctg ggtcttcctc gttgctcttt taagaggtgt ccagtgtcag 60 gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120 tgtgcagcgt ctggattcac cttcagcaac tatggcatgc actgggtccg ccaggctcca 180 ggcaaggggc tggagtgggt ggcaggcatt tggaatgatg gaattaataa ataccatgca 240 cactccgtga ggggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300 caaatgaaca gcccgagagc cgaggacacg gctgtgtatt actgtgcgag agcacggtct 360 ttcgactggc tattatttga gttctggggc cagggaaccc tggtcaccgt ctctagtgcc 420 agcaccaagg ggccatccgt cttccccctg gcgccctgct ccaggagcac ctccgagagc 480 acagccgccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac gaagacctac 660 acctgcaacg tagatcacaa gcccagcaac accaaggtgg acaagagagt tgagtccaaa 720 tatggtcccc catgcccatc atgcccagca cctgagttcc tggggggacc atcagtcttc 780 ctgttccccc caaaacccaa ggacactctc atgatctccc ggacccctga ggtcacgtgc 840 gtggtggtgg acgtgagcca ggaagacccc gaggtccagt tcaactggta cgtggatggc 900 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agttcaacag cacgtaccgt 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctga acggcaagga gtacaagtgc 1020 aaggtctcca acaaaggcct cccgtcctcc atcgagaaaa ccatctccaa agccaaaggg 1080 cagccccgag agccacaggt gtacaccctg cccccatccc aggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 1200 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caggctaacc gtgracaaga gcaggtggca ggaggggaat 1320 gtcttctcat gctccgtgak gcatgaggct ctgcacaacc actacacaca gaagagcctc 1380 tccctgtctc tgggtaaa 1398 <210> 24 <211> 466 <212> PRT <213> Homo sapiens <400> 24 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln             20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe         35 40 45 Ser Asn Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Ala Gly Ile Trp Asn Asp Gly Ile Asn Lys Tyr His Ala 65 70 75 80 His Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Pro Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Ala Arg Ser Phe Asp Trp Leu Leu Phe Glu Phe         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 155 160 Thr Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val     210 215 220 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 225 230 235 240 Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Glu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu         275 280 285 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu     450 455 460 Gly Lys 465 <210> 25 <211> 1407 <212> DNA <213> Homo Sapiens <400> 25 atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60 gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120 tgtgcagtgt ctggattcac cttcagtaac tatggcatgc actgggtccg ccaggctcca 180 ggcaaggggc tggagtgggt ggcagctata tggaatgatg gagaaaataa acaccatgca 240 ggctccgtga ggggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300 caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag aggacgatat 360 tttgactggt tattatttga gtattggggc cagggaaccc tggtcaccgt ctctagtgcc 420 tccaccaagg gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac 660 atctgcaacg tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa 720 tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 780 tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 840 gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 900 gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 960 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1020 tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1080 gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 1140 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1200 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1260 gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1320 caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1380 aagagcctct ccctgtctcc gggtaaa 1407 <210> 26 <211> 469 <212> PRT <213> Homo sapiens <400> 26 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln             20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe         35 40 45 Ser Asn Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Ala Ala Ile Trp Asn Asp Gly Glu Asn Lys His His Ala 65 70 75 80 Gly Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Trp Leu Leu Phe Glu Tyr         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 145 150 155 160 Thr Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val     210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu                 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr             260 265 270 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val         275 280 285 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val     290 295 300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu                 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala             340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro         355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln     370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr                 405 410 415 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu             420 425 430 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser         435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser     450 455 460 Leu Ser Pro Gly Lys 465 <210> 27 <211> 1395 <212> DNA <213> Homo sapiens <400> 27 atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60 gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120 tgtgcagtgt ctggattcac cttcagtaac tatggcatgc actgggtccg ccaggctcca 180 ggcaaggggc tggagtgggt ggcagctata tggaatgatg gagaaaataa acaccatgca 240 ggctccgtga ggggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300 caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag aggacgatat 360 tttgactggt tattatttga gtattggggc cagggaaccc tggtcaccgt ctctagtgcc 420 tccaccaagg gcccatcggt cttccccctg gcgccctgct ccaggagcac ctccgagagc 480 acagcggccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ctctgaccag cggcgtgcac accttcccag ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca acttcggcac ccagacctac 660 acctgcaacg tagatcacaa gcccagcaac accaaggtgg acaagacagt tgagcgcaaa 720 tgttgtgtcg agtgcccacc gtgcccagca ccacctgtgg caggaccgtc agtcttcctc 780 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacgtgcgtg 840 gtggtggacg tgagccacga agaccccgag gtccagttca actggtacgt ggacggcgtg 900 gaggtgcata atgccaagac aaagccacgg gaggagcagt tcaacagcac gttccgtgtg 960 gtcagcgtcc tcaccgttgt gcaccaggac tggctgaacg gcaaggagta caagtgcaag 1020 gtctccaaca aaggcctccc agcccccatc gagaaaacca tctccaaaac caaagggcag 1080 ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag 1140 gtcagcctga cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtgggag 1200 agcaatgggc agccggagaa caactacaag accacacctc ccatgctgga ctccgacggc 1260 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 1320 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc 1380 ctgtctccgg gtaaa 1395 <210> 28 <211> 465 <212> PRT <213> Homo sapiens <400> 28 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln             20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe         35 40 45 Ser Asn Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Ala Ala Ile Trp Asn Asp Gly Glu Asn Lys His His Ala 65 70 75 80 Gly Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Trp Leu Leu Phe Glu Tyr         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 155 160 Thr Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val     210 215 220 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys 225 230 235 240 Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro                 245 250 255 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser             260 265 270 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Ser Glu Asp         275 280 285 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn     290 295 300 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val 305 310 315 320 Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu                 325 330 335 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys             340 345 350 Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr         355 360 365 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr     370 375 380 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 385 390 395 400 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu                 405 410 415 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys             420 425 430 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu         435 440 445 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly     450 455 460 Lys 465 <210> 29 <211> 1398 <212> DNA <213> Homo sapiens <400> 29 atggagtttg ggctgagctg ggttttcctc gttgctcttt taagaggtgt ccagtgtcag 60 gtgcagctgg tggagtctgg gggaggcgtg gtccagcctg ggaggtccct gagactctcc 120 tgtgcagtgt ctggattcac cttcagtaac tatggcatgc actgggtccg ccaggctcca 180 ggcaaggggc tggagtgggt ggcagctata tggaatgatg gagaaaataa acaccatgca 240 ggctccgtga ggggccgatt caccatctcc agagacaatt ccaagaacac gctgtatctg 300 caaatgaaca gcctgagagc cgaggacacg gctgtgtatt actgtgcgag aggacgatat 360 tttgactggt tattatttga gtattggggc cagggaaccc tggtcaccgt ctctagtgcc 420 agcaccaagg ggccatccgt cttccccctg gcgccctgct ccaggagcac ctccgagagc 480 acagccgccc tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg 540 aactcaggcg ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga 600 ctctactccc tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac gaagacctac 660 acctgcaacg tagatcacaa gcccagcaac accaaggtgg acaagagagt tgagtccaaa 720 tatggtcccc catgcccatc atgcccagca cctgagttcc tggggggacc atcagtcttc 780 ctgttccccc caaaacccaa ggacactctc atgatctccc ggacccctga ggtcacgtgc 840 gtggtggtgg acgtgagcca ggaagacccc gaggtccagt tcaactggta cgtggatggc 900 gtggaggtgc ataatgccaa gacaaagccg cgggaggagc agttcaacag cacgtaccgt 960 gtggtcagcg tcctcaccgt cctgcaccag gactggctga acggcaagga gtacaagtgc 1020 aaggtctcca acaaaggcct cccgtcctcc atcgagaaaa ccatctccaa agccaaaggg 1080 cagccccgag agccacaggt gtacaccctg cccccatccc aggaggagat gaccaagaac 1140 caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 1200 gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1260 ggctccttct tcctctacag caggctaacc gtgracaaga gcaggtggca ggaggggaat 1320 gtcttctcat gctccgtgak gcatgaggct ctgcacaacc actacacaca gaagagcctc 1380 tccctgtctc tgggtaaa 1398 <210> 30 <211> 466 <212> PRT <213> Homo sapiens <400> 30 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly 1 5 10 15 Val Gln Cys Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln             20 25 30 Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Phe         35 40 45 Ser Asn Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu     50 55 60 Glu Trp Val Ala Ala Ile Trp Asn Asp Gly Glu Asn Lys His His Ala 65 70 75 80 Gly Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn                 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val             100 105 110 Tyr Tyr Cys Ala Arg Gly Arg Tyr Phe Asp Trp Leu Leu Phe Glu Tyr         115 120 125 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly     130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 155 160 Thr Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val                 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe             180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val         195 200 205 Thr Val Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val     210 215 220 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 225 230 235 240 Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Glu Gly Gly                 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile             260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu         275 280 285 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His     290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys                 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu             340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr         355 360 365 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu     370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val                 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp             420 425 430 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His         435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu     450 455 460 Gly Lys 465 <210> 31 <211> 1401 <212> DNA <213> Homo Sapiens <400> 31 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgag 60 gtgcagctga tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc 120 tgtaagggtt ctggatacag cttttccttc cactggatcg cctgggtgcg ccagatgccc 180 gggaaaggcc tggagtggat ggggatcatc catcctggtg cctctgatac cagatacagc 240 ccgtccttcc aaggccaggt caccatctca gccgacaact ccaacagcgc cacctacctg 300 cagtggagca gcctgaaggc ctcggacacc gccatgtatt tctgtgcgag acaaagggaa 360 ctcgactact ttgactactg gggccaggga accctggtca ccgtctctag tgcctccacc 420 aagggcccat cggtcttccc cctggcaccc tcctccaaga gcacctctgg gggcacagcg 480 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacccagac ctacatctgc 660 aacgtgaatc acaagcccag caacaccaag gtggacaaga aagttgagcc caaatcttgt 720 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 780 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 840 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 900 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 960 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 1020 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 1080 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 1140 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 1200 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1260 gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 1320 aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 1380 ctctccctgt ctccgggtaa a 1401 <210> 32 <211> 467 <212> PRT <213> Homo sapiens <400> 32 Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu Ala Val Leu Gln Gly 1 5 10 15 Val Cys Ala Glu Val Gln Leu Met Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe         35 40 45 Ser Phe His Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu     50 55 60 Glu Trp Met Gly Ile Ile His Pro Gly Ala Ser Asp Thr Arg Tyr Ser 65 70 75 80 Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Asn Ser Asn Ser                 85 90 95 Ala Thr Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met             100 105 110 Tyr Phe Cys Ala Arg Gln Arg Glu Leu Asp Tyr Phe Asp Tyr Trp Gly         115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser     130 135 140 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val                 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala             180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val         195 200 205 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His     210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys 225 230 235 240 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly                 245 250 255 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met             260 265 270 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His         275 280 285 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val     290 295 300 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 305 310 315 320 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly                 325 330 335 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile             340 345 350 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val         355 360 365 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser     370 375 380 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 385 390 395 400 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro                 405 410 415 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val             420 425 430 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met         435 440 445 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser     450 455 460 Pro Gly Lys 465 <210> 33 <211> 1389 <212> DNA <213> Homo sapiens <400> 33 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgag 60 gtgcagctga tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc 120 tgtaagggtt ctggatacag cttttccttc cactggatcg cctgggtgcg ccagatgccc 180 gggaaaggcc tggagtggat ggggatcatc catcctggtg cctctgatac cagatacagc 240 ccgtccttcc aaggccaggt caccatctca gccgacaact ccaacagcgc cacctacctg 300 cagtggagca gcctgaaggc ctcggacacc gccatgtatt tctgtgcgag acaaagggaa 360 ctcgactact ttgactactg gggccaggga accctggtca ccgtctctag tgcctccacc 420 aagggcccat cggtcttccc cctggcgccc tgctccagga gcacctccga gagcacagcg 480 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540 ggcgctctga ccagcggcgt gcacaccttc ccagctgtcc tacagtcctc aggactctac 600 tccctcagca gcgtggtgac cgtgccctcc agcaacttcg gcacccagac ctacacctgc 660 aacgtagatc acaagcccag caacaccaag gtggacaaga cagttgagcg caaatgttgt 720 gtcgagtgcc caccgtgccc agcaccacct gtggcaggac cgtcagtctt cctcttcccc 780 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacgtg cgtggtggtg 840 gacgtgagcc acgaagaccc cgaggtccag ttcaactggt acgtggacgg cgtggaggtg 900 cataatgcca agacaaagcc acgggaggag cagttcaaca gcacgttccg tgtggtcagc 960 gtcctcaccg ttgtgcacca ggactggctg aacggcaagg agtacaagtg caaggtctcc 1020 aacaaaggcc tcccagcccc catcgagaaa accatctcca aaaccaaagg gcagccccga 1080 gaaccacagg tgtacaccct gcccccatcc cgggaggaga tgaccaagaa ccaggtcagc 1140 ctgacctgcc tggtcaaagg cttctacccc agcgacatcg ccgtggagtg ggagagcaat 1200 gggcagccgg agaacaacta caagaccaca cctcccatgc tggactccga cggctccttc 1260 ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1320 tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1380 ccgggtaaa 1389 <210> 34 <211> 463 <212> PRT <213> Homo sapiens <400> 34 Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu Ala Val Leu Gln Gly 1 5 10 15 Val Cys Ala Glu Val Gln Leu Met Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe         35 40 45 Ser Phe His Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu     50 55 60 Glu Trp Met Gly Ile Ile His Pro Gly Ala Ser Asp Thr Arg Tyr Ser 65 70 75 80 Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Asn Ser Asn Ser                 85 90 95 Ala Thr Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met             100 105 110 Tyr Phe Cys Ala Arg Gln Arg Glu Leu Asp Tyr Phe Asp Tyr Trp Gly         115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser     130 135 140 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val                 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala             180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val         195 200 205 Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His     210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys 225 230 235 240 Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val                 245 250 255 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr             260 265 270 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu         275 280 285 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys     290 295 300 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser 305 310 315 320 Val Leu Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys                 325 330 335 Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile             340 345 350 Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro         355 360 365 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu     370 375 380 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 385 390 395 400 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser                 405 410 415 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg             420 425 430 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu         435 440 445 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys     450 455 460 <210> 35 <211> 1392 <212> DNA <213> Homo sapiens <400> 35 atggggtcaa ccgccatcct cgccctcctc ctggctgttc tccaaggagt ctgtgccgag 60 gtgcagctga tgcagtctgg agcagaggtg aaaaagcccg gggagtctct gaagatctcc 120 tgtaagggtt ctggatacag cttttccttc cactggatcg cctgggtgcg ccagatgccc 180 gggaaaggcc tggagtggat ggggatcatc catcctggtg cctctgatac cagatacagc 240 ccgtccttcc aaggccaggt caccatctca gccgacaact ccaacagcgc cacctacctg 300 cagtggagca gcctgaaggc ctcggacacc gccatgtatt tctgtgcgag acaaagggaa 360 ctcgactact ttgactactg gggccaggga accctggtca ccgtctctag tgccagcacc 420 aaggggccat ccgtcttccc cctggcgccc tgctccagga gcacctccga gagcacagcc 480 gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540 ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600 tccctcagca gcgtggtgac cgtgccctcc agcagcttgg gcacgaagac ctacacctgc 660 aacgtagatc acaagcccag caacaccaag gtggacaaga gagttgagtc caaatatggt 720 cccccatgcc catcatgccc agcacctgag ttcctggggg gaccatcagt cttcctgttc 780 cccccaaaac ccaaggacac tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 840 gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 900 gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 960 agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 1020 tccaacaaag gcctcccgtc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 1080 cgagagccac aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtc 1140 agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 1200 aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1260 ttcttcctct acagcaggct aaccgtgrac aagagcaggt ggcaggaggg gaatgtcttc 1320 tcatgctccg tgakgcatga ggctctgcac aaccactaca cacagaagag cctctccctg 1380 tctctgggta aa 1392 <210> 36 <211> 464 <212> PRT <213> Homo sapiens <400> 36 Met Gly Ser Thr Ala Ile Leu Ala Leu Leu Leu Ala Val Leu Gln Gly 1 5 10 15 Val Cys Ala Glu Val Gln Leu Met Gln Ser Gly Ala Glu Val Lys Lys             20 25 30 Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe         35 40 45 Ser Phe His Trp Ile Ala Trp Val Arg Gln Met Pro Gly Lys Gly Leu     50 55 60 Glu Trp Met Gly Ile Ile His Pro Gly Ala Ser Asp Thr Arg Tyr Ser 65 70 75 80 Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Asn Ser Asn Ser                 85 90 95 Ala Thr Tyr Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met             100 105 110 Tyr Phe Cys Ala Arg Gln Arg Glu Leu Asp Tyr Phe Asp Tyr Trp Gly         115 120 125 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser     130 135 140 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 145 150 155 160 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val                 165 170 175 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala             180 185 190 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val         195 200 205 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His     210 215 220 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 225 230 235 240 Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser                 245 250 255 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg             260 265 270 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro         275 280 285 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala     290 295 300 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 305 310 315 320 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr                 325 330 335 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr             340 345 350 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu         355 360 365 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys     370 375 380 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 385 390 395 400 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp                 405 410 415 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser             420 425 430 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala         435 440 445 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys     450 455 460 <210> 37 <211> 705 <212> DNA <213> Homo Sapiens <400> 37 atggaagccc cagctcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60 gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 120 ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 180 ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 240 aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 300 gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctccgct cactttcggc 360 ggagggacca aggtggagat caaacgaact gtggctgcac catctgtctt catcttcccg 420 ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 480 tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 540 caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 600 acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 660 ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 705 <210> 38 <211> 235 <212> PRT <213> Homo sapiens <400> 38 Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser             20 25 30 Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser         35 40 45 Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro     50 55 60 Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala 65 70 75 80 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser                 85 90 95 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser             100 105 110 Asn Trp Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys         115 120 125 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu     130 135 140 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155 160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln                 165 170 175 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser             180 185 190 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu         195 200 205 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser     210 215 220 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 39 <211> 699 <212> DNA <213> Homo Sapiens <400> 39 atgtcgccat cacaactcat tgggtttctg ctgctctggg ttccagcctc caggggtgaa 60 attgtgctga ctcagtctcc agactttcag tctgtgactc caaaggagaa agtcaccatc 120 acctgccggg ccagtcagag cattggtagt agcttacact ggtaccagca gaaaccagat 180 cagtctccaa agctcctcat caagtatgct tcccagtcct tctcaggggt cccctcgagg 240 ttcagtggca gtggatctgg gacagatttc accctcacca tcaatagcct ggaagctgaa 300 gatgctgcag cgtattactg tcatcagagt agtagtttac ctctcacttt cggcggaggg 360 accaaggtgg agatcaaacg aactgtggct gcaccatctg tcttcatctt cccgccatct 420 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 540 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 600 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 660 agctcgcccg tcacaaagag cttcaacagg ggagagtgt 699 <210> 40 <211> 233 <212> PRT <213> Homo sapiens <400> 40 Met Ser Pro Ser Gln Leu Ile Gly Phe Leu Leu Leu Trp Val Pro Ala 1 5 10 15 Ser Arg Gly Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val             20 25 30 Thr Pro Lys Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile         35 40 45 Gly Ser Ser Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys     50 55 60 Leu Leu Ile Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg 65 70 75 80 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser                 85 90 95 Leu Glu Ala Glu Asp Ala Ala Ala Tyr Tyr Cys His Gln Ser Ser Ser             100 105 110 Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr         115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu     130 135 140 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 145 150 155 160 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly                 165 170 175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr             180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His         195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val     210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 <210> 41 <211> 5 <212> PRT <213> Homo sapiens <400> 41 Leu Ser Asp Ile Ala 1 5 <210> 42 <211> 4 <212> PRT <213> Homo sapiens <400> 42 Val Ile Asp Glu One <210> 43 <211> 4 <212> PRT <213> Homo sapiens <400> 43 Thr Cys Phe Ala One <210> 44 <211> 24 <212> DNA <213> Artificial <220> <223> oligonucleotide primer for PCR <220> <221> misc_feature <222> (18). (23) N is a, c, t, or g <400> 44 ggccggatag gcctccannn nnnt 24 <210> 45 <211> 26 <212> DNA <213> artificial <220> <223> oligonucleotide primer for PCR <400> 45 ggacactgac atggactgaa ggagta 26 <210> 46 <211> 26 <212> DNA <213> artificial <220> <223> oligonucleotide primer for PCR <400> 46 ggacactgac atggactgaa ggagta 26 <210> 47 <211> 46 <212> DNA <213> artificial <220> <223> oligonucleotide primer for PCR <400> 47 cagcagaagc ttctagacca ccatgtcgcc atcacaactc attggg 46 <210> 48 <211> 34 <212> DNA <213> artificial <220> <223> oligonucleotide primer for PCR <400> 48 cttgtcgact caacactctc ccctgttgaa gctc 34 <210> 49 <211> 8 <212> PRT <213> artificial <220> <223> amino acid sequence encoded by 5 'anti-IL-1R1 15C4 kappa primer <400> 49 Met Ser Pro Ser Gln Leu Ile Gly 1 5 <210> 50 <211> 7 <212> PRT <213> artificial <220> <223> amino acid sequence encoded by 3 'anti-IL-1R1 15C4 kappa primer <400> 50 Cys Glu Gly Arg Asn Phe Ser 1 5 <210> 51 <211> 1409 <212> DNA <213> Homo sapiens <400> 51 tctagaccac catggacatc aggctcagct tagttttcct tgtccttttc ataaaaggtg 60 tccagtgtga ggtagaactg gtggagtctg ggggcggctt agtacaacct ggaaggtcca 120 tgacactctc ctgtgcagcc tcgggattca ctttcagaac ctatggcatg gcctgggtcc 180 gccaggcccc aacgaagggt ctggagtggg tctcatcaat tactgctagt ggtggtacca 240 cctactatcg agactccgtg aagggccgct tcactatttt tagggataat gcaaaaagta 300 ccctatacct gcagatggac agtccgaggt ctgaggacac ggccacttat ttctgtacat 360 caatttcgga atactggggc cacggagtca tggtcaccgt ctctagtgcc tccaccaagg 420 gcccatcggt cttccccctg gcaccctcct ccaagagcac ctctgggggc acagcggccc 480 tgggctgcct ggtcaaggac tacttccccg aaccggtgac ggtgtcgtgg aactcaggcg 540 ccctgaccag cggcgtgcac accttcccgg ctgtcctaca gtcctcagga ctctactccc 600 tcagcagcgt ggtgaccgtg ccctccagca gcttgggcac ccagacctac atctgcaacg 660 tgaatcacaa gcccagcaac accaaggtgg acaagaaagt tgagcccaaa tcttgtgaca 720 aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg tcagtcttcc 780 tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag gtcacatgcg 840 tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac gtggacggcg 900 tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc acgtaccgtg 960 tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag tacaagtgca 1020 aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa gccaaagggc 1080 agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg accaagaacc 1140 aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc gtggagtggg 1200 agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg gactccgacg 1260 gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag caggggaacg 1320 tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag aagagcctct 1380 ccctgtctcc gggtaaatga taagtcgac 1409 <210> 52 <211> 19 <212> DNA <213> artificial <220> <223> oligonucleotide primer for PCR <400> 52 tgaggacgct gaccacacg 19 <210> 53 <211> 44 <212> DNA <213> artificial <220> <223> oligonucleotide primer for PCR <400> 53 cagcagaagc ttctagacca ccatggggtc aaccgccatc ctcg 44 <210> 54 <211> 32 <212> DNA <213> artificial <220> <223> oligonucleotide primer for PCR <400> 54 gtggaggcac tagagacggt gaccagggtt cc 32 <210> 55 <211> 7 <212> PRT <213> artificial <220> <223> amino acid sequence encoded by 5 'anti-IL-1R1 15C4 heavy chain pr        ime <400> 55 Met Gly Ser Thr Ala Ile Leu 1 5 <210> 56 <211> 11 <212> PRT <213> artificial <220> <223> amino acid sequence encoded by 3 'anti-IL-1R1 15C4 heavy chain pr        ime <400> 56 Thr Ser Ala Ser Ser Val Thr Val Leu Thr Gly 1 5 10 <210> 57 <211> 1415 <212> DNA <213> homo sapiens <400> 57 tctagaccac catggacatg agggtccccg ctcagctcct ggggctcctg ctattgtggt 60 tgagaggtgc cagatgtgag gtccagctgg tgcagtctgg gggaggcttg gtacatcctg 120 gggggtccct gagactctcc tgtgcaggct ctggattcac cttcagtggc catgctttgc 180 actgggttcg ccaggctcca ggaaaaggtc tggagtgggt atcaggtatt ggtactcatg 240 gtgggacata ctatgcagac tccgtgaagg gccgattcac catctccaga gacaatgcca 300 agaactcctt gtttcttcaa atgaacagcc tgagcgccga ggacatggct gtgtattact 360 gtacaagaag aaactgggga caatttgact actggggcca gggaaccctg gtcaccgtct 420 ctagtgcctc caccaagggc ccatcggtct tccccctggc gccctgctcc aggagcacct 480 ccgagagcac agcggccctg ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg 540 tgtcgtggaa ctcaggcgct ctgaccagcg gcgtgcacac cttcccagct gtcctacagt 600 cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcaac ttcggcaccc 660 agacctacac ctgcaacgta gatcacaagc ccagcaacac caaggtggac aagacagttg 720 agcgcaaatg ttgtgtcgag tgcccaccgt gcccagcacc acctgtggca ggaccgtcag 780 tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc cctgaggtca 840 cgtgcgtggt ggtggacgtg agccacgaag accccgaggt ccagttcaac tggtacgtgg 900 acggcgtgga ggtgcataat gccaagacaa agccacggga ggagcagttc aacagcacgt 960 tccgtgtggt cagcgtcctc accgttgtgc accaggactg gctgaacggc aaggagtaca 1020 agtgcaaggt ctccaacaaa ggcctcccag cccccatcga gaaaaccatc tccaaaacca 1080 aagggcagcc ccgagaacca caggtgtaca ccctgccccc atcccgggag gagatgacca 1140 agaaccaggt cagcctgacc tgcctggtca aaggcttcta ccccagcgac atcgccgtgg 1200 agtgggagag caatgggcag ccggagaaca actacaagac cacacctccc atgctggact 1260 ccgacggctc cttcttcctc tacagcaagc tcaccgtgga caagagcagg tggcagcagg 1320 ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac acgcagaaga 1380 gcctctccct gtctccgggt aaatgataag tcgac 1415 <210> 58 <211> 1418 <212> DNA <213> Homo sapiens <400> 58 tctagaccac catggacatg agggtccccg ctcagctcct ggggctcctg ctattgtggt 60 tgagaggtgc cagatgtgag gtccagctgg tgcagtctgg gggaggcttg gtacatcctg 120 gggggtccct gagactctcc tgtgcaggct ctggattcac cttcagtggc catgctttgc 180 actgggttcg ccaggctcca ggaaaaggtc tggagtgggt atcaggtatt ggtactcatg 240 gtgggacata ctatgcagac tccgtgaagg gccgattcac catctccaga gacaatgcca 300 agaactcctt gtttcttcaa atgaacagcc tgagcgccga ggacatggct gtgtattact 360 gtacaagaag aaactgggga caatttgact actggggcca gggaaccctg gtcaccgtct 420 ctagtgccag caccaagggg ccatccgtct tccccctggc gccctgctcc aggagcacct 480 ccgagagcac agccgccctg ggctgcctgg tcaaggacta cttccccgaa ccggtgacgg 540 tgtcgtggaa ctcaggcgcc ctgaccagcg gcgtgcacac cttcccggct gtcctacagt 600 cctcaggact ctactccctc agcagcgtgg tgaccgtgcc ctccagcagc ttgggcacga 660 agacctacac ctgcaacgta gatcacaagc ccagcaacac caaggtggac aagagagttg 720 agtccaaata tggtccccca tgcccatcat gcccagcacc tgagttcctg gggggaccat 780 cagtcttcct gttcccccca aaacccaagg acactctcat gatctcccgg acccctgagg 840 tcacgtgcgt ggtggtggac gtgagccagg aagaccccga ggtccagttc aactggtacg 900 tggatggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag ttcaacagca 960 cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt 1020 acaagtgcaa ggtctccaac aaaggcctcc cgtcctccat cgagaaaacc atctccaaag 1080 ccaaagggca gccccgagag ccacaggtgt acaccctgcc cccatcccag gaggagatga 1140 ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctaccccagc gacatcgccg 1200 tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct cccgtgctgg 1260 actccgacgg ctccttcttc ctctacagca ggctaaccgt ggacaagagc aggtggcagg 1320 aggggaatgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac tacacacaga 1380 agagcctctc cctgtctctg ggtaaatgat aagtcgac 1418 <210> 59 <211> 485 <212> PRT <213> Homo sapiens <400> 59 Met Val His Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly             20 25 30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn         35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr Ala Thr     50 55 60 Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65 70 75 80 Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys Phe                 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile Asp Arg Asn             100 105 110 Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu Arg Ser Ser Val Asn         115 120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile Phe     130 135 140 Thr Arg Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser Leu Leu Glu 145 150 155 160 Ala Asp Lys Cys Lys Glu Arg Glu Glu Lys Ile Ile Leu Val Ser Ser                 165 170 175 Ala Asn Glu Ile Asp Val Arg Pro Cys Pro Leu Asn Pro Asn Glu His             180 185 190 Lys Gly Thr Ile Thr Trp Tyr Lys Asp Asp Ser Lys Thr Pro Val Ser         195 200 205 Thr Glu Gln Ala Ser Arg Ile His Gln His Lys Glu Lys Leu Trp Phe     210 215 220 Val Pro Ala Met Val Glu Asp Ser Gly His Tyr Tyr Cys Val Val Arg 225 230 235 240 Asn Ser Ser Tyr Cys Leu Arg Ile Lys Ile Ser Ala Lys Phe Val Glu                 245 250 255 Asn Glu Pro Asn Leu Cys Tyr Asn Ala Gln Ala Ile Phe Lys Gln Lys             260 265 270 Leu Pro Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr Met Glu Phe         275 280 285 Phe Lys Asn Glu Asn Asn Glu Leu Pro Lys Leu Gln Trp Tyr Lys Asp     290 295 300 Cys Lys Pro Leu Leu Leu Asp Asn Ile His Phe Ser Gly Val Lys Asp 305 310 315 320 Arg Leu Ile Val Met Asn Val Ala Glu Lys His Arg Gly Asn Tyr Thr                 325 330 335 Cys His Ala Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr Pro Ile Thr Arg             340 345 350 Val Ile Glu Phe Ile Thr Leu Glu Glu Asn Lys Pro Thr Arg Pro Val         355 360 365 Ile Val Ser Pro Ala Asn Glu Thr Met Glu Val Asp Leu Gly Ser Gln     370 375 380 Ile Gln Leu Ile Cys Asn Val Thr Gly Gln Leu Ser Asp Ile Ala Tyr 385 390 395 400 Trp Lys Trp Asn Gly Ser Val Ile Asp Glu Asp Asp Pro Val Leu Gly                 405 410 415 Glu Asp Tyr Tyr Ser Val Glu Asn Pro Ala Asn Lys Arg Arg Ser Thr             420 425 430 Leu Ile Thr Val Leu Asn Ile Ser Glu Ile Glu Ser Arg Phe Tyr Lys         435 440 445 His Pro Phe Thr Cys Phe Ala Lys Asn Thr His Gly Ile Asp Ala Ala     450 455 460 Tyr Ile Gln Leu Ile Tyr Pro Val Thr Asn Phe Gln Lys Asp Tyr Lys 465 470 475 480 Asp Asp Asp Asp Lys                 485 <210> 60 <211> 485 <212> PRT <213> artificial <220> <223> avidin-cynomolgus IL-1R1-FLAG chimeric protein <400> 60 Met Val His Ala Thr Ser Pro Leu Leu Leu Leu Leu Leu Leu Ser Leu 1 5 10 15 Ala Leu Val Ala Pro Gly Leu Ser Ala Arg Lys Cys Ser Leu Thr Gly             20 25 30 Lys Trp Thr Asn Asp Leu Gly Ser Asn Met Thr Ile Gly Ala Val Asn         35 40 45 Ser Lys Gly Glu Phe Thr Gly Thr Tyr Thr Thr Ala Val Thr Ala Thr     50 55 60 Ser Asn Glu Ile Lys Glu Ser Pro Leu His Gly Thr Gln Asn Thr Ile 65 70 75 80 Asn Lys Arg Thr Gln Pro Thr Phe Gly Phe Thr Val Asn Trp Lys Phe                 85 90 95 Ser Glu Ser Thr Thr Val Phe Thr Gly Gln Cys Phe Ile Asp Arg Asn             100 105 110 Gly Lys Glu Val Leu Lys Thr Met Trp Leu Leu Arg Ser Ser Val Asn         115 120 125 Asp Ile Gly Asp Asp Trp Lys Ala Thr Arg Val Gly Ile Asn Ile Phe     130 135 140 Thr Arg Leu Arg Thr Gln Lys Glu Gln Leu Leu Ala Ser Leu Leu Glu 145 150 155 160 Ala Asp Lys Cys Asn Glu Arg Glu Glu Lys Ile Ile Leu Val Ser Ser                 165 170 175 Ala Asn Glu Ile Asp Val Arg Pro Cys Pro Leu Asn Pro Asn Glu Tyr             180 185 190 Lys Gly Thr Ile Thr Trp Tyr Lys Asn Asp Ser Lys Thr Pro Ile Ser         195 200 205 Thr Glu Gln Ala Ser Arg Ile His Gln His Lys Lys Lys Leu Trp Phe     210 215 220 Val Pro Ala Lys Val Glu Asp Ser Gly His Tyr Tyr Cys Val Val Arg 225 230 235 240 Asn Ser Ser Tyr Cys Leu Arg Ile Lys Ile Thr Ala Lys Phe Val Glu                 245 250 255 Asn Glu Pro Asn Leu Cys Tyr Asn Ala Glu Ala Ile Phe Lys Gln Arg             260 265 270 Leu Pro Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr Met Glu Phe         275 280 285 Phe Lys Asp Glu Asn Asn Glu Leu Pro Lys Leu Leu Trp Tyr Lys Asp     290 295 300 Cys Lys Pro Leu Leu Leu Asp Asn Ile His Phe Ser Gly Val Lys Asp 305 310 315 320 Arg Leu Ile Val Met Asn Val Ala Glu Lys His Arg Gly Asn Tyr Thr                 325 330 335 Cys His Ala Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr Pro Ile Thr Arg             340 345 350 Val Ile Glu Phe Ile Thr Leu Glu Glu Asn Lys Pro Thr Arg Pro Val         355 360 365 Ile Val Ser Pro Ala Asn Glu Thr Ile Glu Val Asp Leu Gly Ser Gln     370 375 380 Ile Gln Leu Ile Cys Asn Val Thr Gly Gln Leu Ser Asp Thr Ala Tyr 385 390 395 400 Trp Lys Trp Asn Gly Ser Phe Ile Asp Glu Asp Asp Pro Val Leu Gly                 405 410 415 Glu Asp Tyr Tyr Ser Val Glu Asn Pro Ala Asn Lys Arg Arg Ser Thr             420 425 430 Leu Ile Thr Val Leu Asn Ile Ser Glu Thr Glu Ser Arg Phe Tyr Lys         435 440 445 His Pro Phe Thr Cys Leu Ala Arg Asn Thr His Gly Met Asp Ala Ala     450 455 460 Tyr Val Gln Leu Ile Tyr Pro Val Thr Lys Phe Gln Lys Asp Tyr Lys 465 470 475 480 Asp Asp Asp Asp Lys                 485 <210> 61 <211> 5 <212> PRT <213> Homo sapiens <400> 61 Asn Tyr Gly Met His 1 5 <210> 62 <211> 8 <212> PRT <213> Homo sapiens <400> 62 Thr Phe Ser Asn Tyr Gly Met His 1 5 <210> 63 <211> 5 <212> PRT <213> Homo sapiens <400> 63 Phe His Trp Ile Ala 1 5 <210> 64 <211> 17 <212> PRT <213> Homo sapiens <400> 64 Gly Ile Trp Asn Asp Gly Ile Asn Lys Tyr His Ala His Ser Val Arg 1 5 10 15 Gly      <210> 65 <211> 17 <212> PRT <213> Homo sapiens <400> 65 Ala Ile Trp Asn Asp Gly Glu Asn Lys His His Ala Gly Ser Val Arg 1 5 10 15 Gly      <210> 66 <211> 17 <212> PRT <213> Homo sapiens <400> 66 Ile Ile His Pro Gly Ala Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln 1 5 10 15 Gly      <210> 67 <211> 11 <212> PRT <213> Homo sapiens <400> 67 Ala Arg Ser Phe Asp Trp Leu Leu Phe Glu Phe 1 5 10 <210> 68 <211> 11 <212> PRT <213> Homo sapiens <400> 68 Gly Arg Tyr Phe Asp Trp Leu Leu Phe Glu Tyr 1 5 10 <210> 69 <211> 9 <212> PRT <213> Homo sapiens <400> 69 Gln Arg Glu Leu Asp Tyr Phe Asp Tyr 1 5 <210> 70 <211> 11 <212> PRT <213> Homo sapiens <400> 70 Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala 1 5 10 <210> 71 <211> 11 <212> PRT <213> Homo sapiens <400> 71 Arg Ala Ser Gln Ser Ile Gly Ser Ser Leu His 1 5 10 <210> 72 <211> 7 <212> PRT <213> Homo sapiens <400> 72 Asp Ala Ser Asn Arg Ala Thr 1 5 <210> 73 <211> 7 <212> PRT <213> Homo sapiens <400> 73 Tyr Ala Ser Gln Ser Phe Ser 1 5 <210> 74 <211> 10 <212> PRT <213> Homo sapiens <400> 74 Gln Gln Arg Ser Asn Trp Pro Pro Leu Thr 1 5 10 <210> 75 <211> 9 <212> PRT <213> Homo sapiens <400> 75 His Gln Ser Ser Ser Leu Pro Leu Thr 1 5 <210> 76 <211> 111 <212> PRT <213> Homo sapiens <400> 76 Pro Val Ile Val Ser Pro Ala Asn Glu Thr Met Glu Val Asp Leu Gly 1 5 10 15 Ser Gln Ile Gln Leu Ile Cys Asn Val Thr Gly Gln Leu Ser Asp Ile             20 25 30 Ala Tyr Trp Lys Trp Asn Gly Ser Val Ile Asp Glu Asp Asp Pro Val         35 40 45 Leu Gly Glu Asp Tyr Tyr Ser Val Glu Asn Pro Ala Asn Lys Arg Arg     50 55 60 Ser Thr Leu Ile Thr Val Leu Asn Ile Ser Glu Ile Glu Ser Arg Phe 65 70 75 80 Tyr Lys His Pro Phe Thr Cys Phe Ala Lys Asn Thr His Gly Ile Asp                 85 90 95 Ala Ala Tyr Ile Gln Leu Ile Tyr Pro Val Thr Asn Phe Gln Lys             100 105 110 <210> 77 <211> 350 <212> DNA <213> Homo sapiens <400> 77 cctgtgattg tgagcccagc taatgagaca atggaagtag acttgggatc ccagatacaa 60 ttgatctgta atgtcaccgg ccagttgagt gacattgctt actggaagtg gaatgggtca 120 gtaattgatg aagatgaccc agtgctaggg gaagactatt acagtgtgga aaatcctgca 180 aacaaaagaa ggagtaccct catcacagtg cttaatatat cggaaattga aagtagattt 240 tataaacatc catttacctg ttttgccaag aatacacatg gtatagatgc agcatatatc 300 cagttaatat atccagtcac taatttccag aagcacatga ttggtatatg 350 <210> 78 <211> 350 <212> DNA <213> Rattus sp. <400> 78 cctgtgatta tgagcccacg gaatgagacg atggaagctg acccaggatc cacgatacaa 60 ctgatctgca acgtcacggg ccagttcacc gaccttgtct actggaagtg gaatgggtcg 120 gaaattgaat gggacgatcc aatcctagcc gaagactatc agtttttgga acacccttca 180 gccaaaagaa agtacactct cattacaaca cttaacgttt cagaggtcaa aagccagttt 240 tatcgctatc cgttcatctg cttcgttaag aacactcata ttctggagac tgcacacgta 300 cggttagtat acccagttcc tgacttcaag aattacctca tcgggggctt 350 <210> 79 <211> 111 <212> PRT <213> Rattus sp. <400> 79 Pro Val Ile Met Ser Pro Arg Asn Glu Thr Met Glu Ala Asp Pro Gly 1 5 10 15 Ser Thr Ile Gln Leu Ile Cys Asn Val Thr Gly Gln Phe Thr Asp Leu             20 25 30 Val Tyr Trp Lys Trp Asn Gly Ser Glu Ile Glu Trp Asp Asp Pro Ile         35 40 45 Leu Ala Glu Asp Tyr Gln Phe Leu Glu His Pro Ser Ala Lys Arg Lys     50 55 60 Tyr Thr Leu Ile Thr Thr Leu Asn Val Ser Glu Val Lys Ser Gln Phe 65 70 75 80 Tyr Arg Tyr Pro Phe Ile Cys Phe Val Lys Asn Thr His Ile Leu Glu                 85 90 95 Thr Ala His Val Arg Leu Val Tyr Pro Val Pro Asp Phe Lys Lys             100 105 110

Claims (67)

delete delete delete delete An isolated human antibody that specifically binds to interleukin-1 receptor type 1 (IL-1R1), including (a) consists of the amino acid sequence set forth in SEQ ID NO: 10 or     Or has a heavy chain variable region comprising an antigen-binding fragment thereof     Heavy chain, and     Or consists of the amino acid sequence set forth in SEQ ID NO: 12     Or has a light chain variable region comprising an antigen-binding fragment thereof     Light chain; (b) a heavy chain consisting of an amino acid sequence set forth in SEQ ID NO: 14 or having a heavy chain variable region comprising an antigen-binding fragment thereof, and    A light chain consisting of an amino acid sequence set forth in SEQ ID NO: 12 or having a light chain variable region comprising an antigen-binding fragment thereof; or (c) a heavy chain consisting of an amino acid sequence set forth in SEQ ID NO: 16 or having a heavy chain variable region comprising an antigen-binding fragment thereof, and    Consists of the amino acid sequence set forth in SEQ ID NO: 18, or    Or has a light chain variable region comprising an antigen-binding fragment thereof    Light chain. 6. The heavy chain of claim 5, wherein the heavy chain comprises a heavy chain variable region consisting of or comprising an amino acid sequence set forth in SEQ ID NO: 10 and comprising an antigen-binding fragment thereof, and the light chain is an amino acid set forth in SEQ ID NO: 12 An antibody comprising a light chain variable region consisting of a sequence or comprising an antigen-binding fragment thereof. delete 6. The heavy chain of claim 5, wherein the heavy chain comprises a heavy chain variable region consisting of or comprising an antigen-binding fragment thereof as set forth in SEQ ID NO: 14, and the light chain as set forth in SEQ ID NO: 12 An antibody, characterized in that it comprises a light chain variable region consisting of or comprising an antigen-binding fragment thereof. delete The heavy chain of claim 5, wherein the heavy chain comprises a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 16 or comprising an antigen-binding fragment thereof, and the light chain is an amino acid set forth in SEQ ID NO: 18 An antibody, characterized in that it comprises a light chain variable region consisting of or comprising an antigen-binding fragment thereof. delete An isolated human antibody that specifically binds to interleukin-1 receptor type 1 (IL-1R1), including (a) consists of the amino acid sequence set forth in SEQ ID NO: 38 or     Or a light chain comprising an antigen-binding fragment thereof; And (b) SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26,     Amino acids set forth in SEQ ID NO: 28 or SEQ ID NO: 30     Consisting of or comprising antigen-binding fragments thereof     Heavy chain. 13. The heavy chain of claim 12, wherein the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 20 or comprises an antigen-binding fragment thereof, and the light chain consists of the amino acid sequence set forth in SEQ ID NO: 38 or An antibody, characterized in that it comprises an antigen-binding fragment. delete The method of claim 12, wherein the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 22 or comprises an antigen-binding fragment thereof, and the light chain consists of the amino acid sequence set forth in SEQ ID NO: 38 or An antibody, characterized in that it comprises an antigen-binding fragment. delete The compound of claim 12, wherein the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 24 or comprises an antigen-binding fragment thereof, and the light chain consists of the amino acid sequence set forth in SEQ ID NO: 38 or An antibody, characterized in that it comprises an antigen-binding fragment. delete The compound of claim 12, wherein the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 26 or comprises an antigen-binding fragment thereof, and the light chain consists of the amino acid sequence set forth in SEQ ID NO: 38 or An antibody, characterized in that it comprises an antigen-binding fragment. delete 13. The heavy chain of claim 12, wherein the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 28 or comprises an antigen-binding fragment thereof, and the light chain consists of the amino acid sequence set forth in SEQ ID NO: 38 or An antibody, characterized in that it comprises an antigen-binding fragment. delete 13. The heavy chain of claim 12, wherein the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 30 or comprises an antigen-binding fragment thereof, and the light chain consists of the amino acid sequence set forth in SEQ ID NO: 38 or An antibody, characterized in that it comprises an antigen-binding fragment. delete An isolated human antibody that specifically binds to interleukin-1 receptor type 1 (IL-1R1), including (a) consists of the amino acid sequence set forth in SEQ ID NO: 40 or     Or a light chain comprising an antigen-binding fragment thereof; And (b) described in SEQ ID NO: 32, SEQ ID NO: 34 or SEQ ID NO: 36     Consisting of an amino acid sequence or antigen-binding fragment thereof     Heavy chain containing. The composition of claim 25, wherein the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 32 or comprises an antigen-binding fragment thereof, and the light chain consists of the amino acid sequence set forth in SEQ ID NO: 40 or An antibody, characterized in that it comprises an antigen-binding fragment. delete The composition of claim 25, wherein the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 34 or comprises an antigen-binding fragment thereof, and the light chain consists of the amino acid sequence set forth in SEQ ID NO: 40 or An antibody, characterized in that it comprises an antigen-binding fragment. delete The composition of claim 25, wherein the heavy chain consists of the amino acid sequence set forth in SEQ ID NO: 36 or comprises an antigen-binding fragment thereof, and the light chain consists of the amino acid sequence set forth in SEQ ID NO: 40 or An antibody, characterized in that it comprises an antigen-binding fragment. delete 26. The antibody of claim 5, 12 or 25, wherein the heavy and light chains are linked by a flexible linker to form a single chain of antibody. 33. The antibody of claim 32 which is a single chain Fv antibody. 26. The antibody of claim 5, 12 or 25, which is a Fab. 26. The antibody of claim 5, 12 or 25, which is Fab '. 26. The antibody of claim 5, 12 or 25, which is (Fab ') ₂ . 26. The antibody of claim 5, 12 or 25, wherein the antibody is a fully human antibody. 26. The antibody of claim 5, 12 or 25, wherein the antibody inhibits the binding of IL-1 to the receptor. delete A pharmaceutical composition for treating an IL-1 mediated disease selected from the following comprising a therapeutically effective amount of the antibody of claim 38 and a pharmaceutically acceptable carrier. The IL-1 mediated disease is acute pancreatitis; Amyotrophic Lateral Sclerosis (ALS); Alzheimer's disease; Cachexia / anorexia, including AIDS-induced cachexia; asthma ; Atherosclerosis; Autoimmune vasculitis; Chronic fatigue syndrome; Clostridium related diseases, including Clostridium-related diarrhea; Coronary symptoms and signs, including congestive heart failure, coronary restenosis, myocardial infarction, myocardial dysfunction, and coronary bypass grafts; Tumors, as well as cancer, multiple myeloma and myeloid leukemia and other leukemias; diabetes ; Endometriosis; Fever; Fibromyalgia; Glomerulonephritis; Large host graft disease / transplant rejection; Hemorrhagic shock; Hyperalgesia; Inflammatory bowel disease; Inflammatory symptoms of joints, including osteoarthritis, psoriatic arthritis and rheumatoid arthritis; Inflammatory eye disease; Ischemia including cerebral ischemia; Kawasaki disease; Learning disability; Lung disease; Multiple sclerosis; Myopathy; Neurotoxicity; osteoporosis ; Pain, including cancer-related pain; Parkinson's disease; Periodontal periodontal disease; Early delivery; Psoriasis; Reperfusion injury; Sepsis 쇽; Side effects from radiation therapy; Temporomandibular joint disease; Sleep disorder; Uveitis; And inflammatory symptoms due to contusion, sprain, cartilage damage, trauma, orthopedic surgery, infection or other disease progression. delete A heavy chain consisting of a variable region consisting of or comprising an amino acid sequence set forth in any one of SEQ ID NO: 10, SEQ ID NO: 14 and SEQ ID NO: 16, or comprising an antigen-binding fragment thereof, and a constant region. SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34 and SEQ ID NO: heavy chain consisting of the amino acid sequence set forth in any one of 36 or comprising an antigen-binding fragment thereof. A light chain comprising a variable region consisting of or comprising an amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 18, or comprising an antigen-binding fragment thereof, and a constant region. A light chain consisting of or comprising an antigen-binding fragment thereof of the amino acid sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 40. Isolated human antibody comprising (a) human heavy chain CDR1 domain consisting of the amino acid sequence of the human heavy chain outline region, SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63     Reverse, amino of SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66     Human heavy chain CDR2 region consisting of an acid sequence, and SEQ ID NO: 67,     Consisting of the amino acid sequence of SEQ ID NO: 68 or SEQ ID NO: 69     Human heavy chain CDR3 region; And (b) the human light chain outline region, SEQ ID NO: 70 or SEQ ID NO: 71     Human light chain CDR1 region consisting of the amino acid sequence, SEQ ID NO: 72     Or a human light chain consisting of the amino acid sequence of SEQ ID NO: 73     The CDR2 region and amino acid of SEQ ID NO: 74 or SEQ ID NO: 75     Human light chain CDR3 region consisting of sequences Here, the antibody specifically binds to interleukin-1 receptor type 1 (IL-1R1). delete delete delete delete delete delete delete delete delete delete 26. The antibody of claim 5, 12 or 25, which is an IgG2 antibody. 26. The antibody of claim 5, 12 or 25, characterized in that it specifically binds to a polypeptide of SEQ ID NO: 76. 26. The antibody of claim 5, 12 or 25, wherein the antibody binds specifically to epitope 4 comprising the amino acid sequence YSV of IL-1R1. delete delete With an amino acid sequence of SEQ ID NO: 61, SEQ ID NO: 62 or SEQ ID NO: 63, which forms part of a human monoclonal antibody that specifically binds to Interleukin-1 receptor type 1 (IL-1R1) Heavy chain variable domain CDR1. With an amino acid sequence of SEQ ID NO: 64, SEQ ID NO: 65 or SEQ ID NO: 66, which forms part of a human monoclonal antibody that specifically binds to Interleukin-1 receptor type 1 (IL-1R1) Heavy chain variable domain CDR2. With an amino acid sequence of SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69, which forms part of a human monoclonal antibody that specifically binds to Interleukin-1 receptor type 1 (IL-1R1) Heavy chain variable domain CDR3. A light chain variable domain CDR1 consisting of the amino acid sequence of SEQ ID NO: 70 or SEQ ID NO: 71, which forms part of a human monoclonal antibody that specifically binds to interleukin-1 receptor type 1 (IL-1R1). A light chain variable domain CDR2 consisting of the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 73, which forms part of a human monoclonal antibody that specifically binds to Interleukin-1 receptor type 1 (IL-1R1). A light chain variable domain CDR3 consisting of the amino acid sequence of SEQ ID NO: 74 or SEQ ID NO: 75, which forms part of a human monoclonal antibody that specifically binds to interleukin-1 receptor type 1 (IL-1R1).

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